Real-time focusing in line scan imaging

ABSTRACT

System for acquiring a digital image of a sample on a microscope slide. In an embodiment, the system comprises a stage configured to support a sample, an objective lens having a single optical axis that is orthogonal to the stage, an imaging sensor, and a focusing sensor. The system further comprises at least one beam splitter optically coupled to the objective lens and configured to receive a field of view corresponding to the optical axis of the objective lens, and simultaneously provide at least a first portion of the field of view to the imaging sensor and at least a second portion of the field of view to the focusing sensor. The focusing sensor may simultaneously acquire image(s) at a plurality of different focal distances and/or simultaneously acquire a pair of mirrored images, each comprising pixels acquired at a plurality of different focal distances.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/763,061, filed on Mar. 23, 2018, which is a national stage entry ofInternational Patent App. No. PCT/US2016/053581, filed on Sep. 23, 2016,which claims priority to U.S. Provisional Patent App. No. 62/232,229,filed on Sep. 24, 2015, which are both hereby incorporated herein byreference as if set forth in full.

This application is related to U.S. patent application Ser. No.14/398,443, filed on Oct. 31, 2014, which claims priority toInternational Patent App. No. PCT/US2013/031045, filed on Mar. 13, 2013,and U.S. Provisional Application No. 61/641,788, filed on May 2, 2012,which are all hereby incorporated herein by reference as if set forth infull.

BACKGROUND Field of the Invention

The present invention generally relates to digital pathology and moreparticularly relates to a multiple independent linear sensor apparatusfor performing real-time focusing in line scan imaging.

Related Art

Most auto-focus methods in microscope imaging systems can be dividedinto two categories: laser-based interferometer for sensing slideposition, and image content analysis. With laser-based interferometermethods, measuring the reflection of the laser beam off a slide surfacecan provide only global focus information of the slide position or thecover slip position. It lacks focusing accuracy for tissue samples withlarge height variations. In addition, the image content analysis methodsrequire multiple image acquisitions at different focus depths, and usealgorithms to compare the images to determine the best focus. However,acquiring multiple images at different focus depths may create timedelays between focusing and imaging. Therefore, what is needed is asystem and method that overcomes these significant problems found in theconventional methods described above.

SUMMARY

In an embodiment, a system for scanning a sample to acquire a digitalimage of the sample is disclosed. The system may comprise: a stageconfigured to support a sample; an objective lens having a singleoptical axis that is orthogonal to the stage; an imaging sensor; afocusing sensor; and at least one first beam splitter optically coupledto the objective lens and configured to receive a field of viewcorresponding to the optical axis of the objective lens, andsimultaneously provide at least a first portion of the field of view tothe imaging sensor and at least a second portion of the field of view tothe focusing sensor.

The focusing sensor may receive the second portion of the field of viewalong an optical path, wherein the focusing sensor is tilted at an anglewith respect to the optical path, such that the second portion of thefield of view is acquired, by the focusing sensor, as an imagecomprising pixels representing different focal distances. Furthermore,the focusing sensor may comprise a plurality of regions, wherein eachregion of the focusing sensor receives the second portion of the fieldof view along a separate optical path, and wherein the focusing sensoris tilted at an angle with respect to each of the separate opticalpaths, such that the second portion of the field of view is acquired, byeach region of focusing sensor, at a different focal distance than theother regions of focusing sensor.

Alternatively, the focusing sensor may comprise a plurality of regions,wherein each region of the focusing sensor receives the second portionof the field of view along a separate optical path, wherein the focusingsensor is orthogonal with respect to each of the separate optical paths,and wherein each of the separate optical paths has a different focusdistance, such that the second portion of the field of view is acquired,by each region of focusing sensor, at a different focal distance thanthe other regions of focusing sensor.

Alternatively, the focusing sensor may comprise a first portion and asecond portion, wherein the first portion of the focusing sensorreceives the second portion of the field of view along a first opticalpath and is tilted at a first angle with respect to the first opticalpath, and wherein the second portion of the focusing sensor receives thesecond portion of the field of view along a second optical path, that isseparate from the first optical path, and is tilted at a second anglewith respect to the second optical path that is reverse to the firstangle.

Alternatively, the focusing sensor may comprise a first region and asecond region, wherein the first region receives the second portion ofthe field of view along a first optical path, wherein the second regionreceives a mirrored second portion of the field of view along a secondoptical path, and wherein the focusing sensor is tilted at an angle withrespect to each of the first optical path and the second optical path.

Alternatively, the tilted sensor can be substituted with a non-tiltedsensor and a wedge prism placed in front of the non-tilted sensor. Theangle of the tilt can have a negative value, a zero value, or a positivevalue.

Alternatively, the focusing sensor may be a section of a sensor with awedge optic in front of it to create focal variation covering thisfocusing section of the sensor along the sensor axis, while the othersection of the sensor acts as an imaging sensor.

In addition, the system may comprise a processor that is configured to,for each portion of the sample to be scanned: acquire a focusing imageof the portion of the sample from the focusing sensor; for each of aplurality of positions on the focusing sensor, calculate a contrastmeasure for a region of the focusing image corresponding to thatposition on the focusing sensor; determine a peak for the contrastmeasures; and determine a position for the objective lens that providesthe peak for the contrast measures at the one parfocal point on thefocusing sensor.

In another embodiment, a method for automatic real-time focusing isdisclosed. The method may comprise using a processor in a slide scannerto, for each portion of a sample to be scanned: prior to, at the sametime as, or after the portion of the sample being sensed by an imagingsensor, acquire a focusing image of the portion of the sample from afocusing sensor, wherein a portion of a field of view that is sensed bythe focusing sensor is offset from a portion of the field of view thatis sensed by the imaging sensor, such that, in a scan direction, thefocusing sensor senses a portion of the field of view before, at thetime that, or after the imaging sensor senses that same portion of thefield of view, and wherein one point on the focusing sensor is parfocalwith the imaging sensor, for each a plurality of positions on thefocusing sensor, calculate a contrast measure for a region of thefocusing image corresponding to that position on the focusing sensor,determine a peak for the contrast measures, determine a position for anobjective lens that provides the peak for the contrast measures at theparfocal point on the focusing sensor, and move the objective lens tothe determined position; and acquire an image of the portion of thesample from the imaging sensor while the objective lens is at thedetermined position.

In another embodiment, a non-transitory computer-readable medium havinginstructions stored thereon is disclosed. The instructions, whenexecuted by a processor, cause the processor to, for each portion of asample to be scanned: prior to, at the same time as, or after theportion of the sample being sensed by an imaging sensor, acquire afocusing image of the portion of the sample from a focusing sensor,wherein a portion of a field of view that is sensed by the focusingsensor is offset from a portion of the field of view that is sensed bythe imaging sensor, such that, in a scan direction, the focusing sensorsenses a portion of the field of view before, at the same time that, orafter the imaging sensor senses that same portion of the field of view,and wherein one point on the is parfocal with the imaging sensor, foreach a plurality of positions on the focusing sensor, calculate acontrast measure for a region of the focusing image corresponding tothat position on the focusing sensor, determine a peak for the contrastmeasures, determine a position for an objective lens that provides thepeak for the contrast measures at the parfocal point on the focusingsensor, and move the objective lens to the determined position; andacquire an image of the portion of the sample from the imaging sensorwhile the objective lens is at the determined position.

In another embodiment, a relationship (e.g., a difference or a ratio)between the contrast measure from the focusing image and the contrastmeasure from the main image is defined, and the peak of thisrelationship is determined, to thereby determine the position of theobjective lens with respect to the parfocal point.

Other features and advantages of the disclosed embodiments will becomereadily apparent to those of ordinary skill in the art after reviewingthe following detailed description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The structure and operation of embodiments will be understood from areview of the following detailed description and the accompanyingdrawings in which like reference numerals refer to like parts and inwhich:

FIG. 1 is a block diagram illustrating an example side viewconfiguration of a scanning system, according to an embodiment;

FIG. 2 is a block diagram illustrating an example configuration of afocusing sensor and imaging sensor with respect to a radius ofillumination and a circular optical field of view, according to anembodiment;

FIG. 3A is a block diagram illustrating an example top viewconfiguration of a imaging sensor, according to an embodiment;

FIG. 3B is a block diagram illustrating an example top viewconfiguration of a tilted focusing sensor, according to an embodiment;

FIG. 3C is a block diagram illustrating an example of a sensor, in whichhalf of the sensor is used to produce a normal image, and the other halfis used to produce an image with various focal depths across it,according to an embodiment;

FIG. 4 is a block diagram illustrating an example top view configurationof a tilted focusing sensor, according to an embodiment;

FIG. 5 is a time chart diagram illustrating an example interplay betweena focusing sensor and an imaging sensor during scanning, according to anembodiment;

FIG. 6 is a block diagram illustrating an example tilted focusing sensorwith focusing optics, according to an embodiment;

FIGS. 7A-7C are block diagrams illustrating an example non-tiltedfocusing sensor with focusing optics, according to an embodiment;

FIG. 8 illustrates example results from a peak-finding algorithm,according to an embodiment.

FIG. 9A illustrates a focal relationship between a tilted focusingsensor and imaging sensor, according to an embodiment.

FIGS. 9B-9D illustrate the relationships of contrast functions for atilted focusing sensor and imaging sensor, according to an embodiment.

FIG. 10 illustrates an example tilted focusing sensor comprising twotilted line sensors, according to an embodiment.

FIG. 11A is a block diagram illustrating an example tilted focusingsensor with focusing optics for acquiring reversed images, according toan embodiment.

FIG. 11B is block diagram illustrating an example tilted focusing sensorwith focusing optics for acquiring reversed images, according to anembodiment.

FIG. 12A illustrates the directionality of focal distances for twoimages acquired by a focusing sensor, according to an embodiment.

FIG. 12B illustrates contrast functions for two reversed images acquiredby a focusing sensor, according to an embodiment.

FIG. 13 is a flow diagram of a real-time focusing process, according toan embodiment.

FIG. 14A is a block diagram illustrating an example microscope slidescanner, according to an embodiment;

FIG. 14B is a block diagram illustrating an alternative examplemicroscope slide scanner, according to an embodiment;

FIG. 14C is a block diagram illustrating example linear sensor arrays,according to an embodiment; and

FIG. 15 is a block diagram illustrating an example wired or wirelessprocessor-enabled device that may be used in connection with variousembodiments described herein.

DETAILED DESCRIPTION

Certain embodiments are based on image content analysis (e.g., tissuefinding and macro focus), and take advantage of line imaging and linefocusing for accurate real-time auto-focusing. In one embodiment, fullstripe focusing is performed during a retrace process of line scanning.In an alternative embodiment, focusing is performed during imagescanning. Both embodiments eliminate time delays in image scanning,thereby speeding up the entire digital image scanning process. Inaddition, certain embodiments provide for real-time (i.e., instantaneousor near-instantaneous) focusing in line scan imaging using multiplelinear detectors or other components. After reading this description itwill become apparent to one skilled in the art how to implement variousalternative embodiments and use those embodiments in alternativeapplications. However, although various embodiments will be describedherein, it is understood that these embodiments are presented by way ofexample only, and not limitation. As such, this detailed description ofvarious alternative embodiments should not be construed to limit thescope or breadth of the present application as set forth in the appendedclaims.

In an embodiment, one or more focus points are determined for a sample(e.g., a tissue sample prepared on a glass microscope slide). Forexample, a macro focus point or a plurality of focus points may bedetermined for the sample. Specifically, one or more positions on thesample may be determined. For each of these positions, the sample may bemoved along X and Y axes (e.g., by a motorized stage), such that, thatposition on the sample is located under an objective lens. In analternative embodiment, the objective lens may be moved along X and Yaxes, or both the objective lens and the sample may be moved along X andY axes, such that the objective lens is located above each position onthe sample. In any case, for each position, an image of the region ofthe sample at that position may be acquired at a plurality of focusheights, while the sample is stationary in the X and Y axes, via afocusing sensor optically coupled with the objective lens as theobjective lens is moved along a Z axis (i.e., orthogonal to both the Xand Y axes) through the plurality of focus heights. Software may be usedto compute the best focus height for each position, based on the imagesacquired at the plurality of focus heights for the position. A real-timefocus mechanism may then constrain the objective lens at the computedbest focus heights at the corresponding positions, via a feedback loop,while scanning the entire sample. It should be understood that while theterm “focus height” or “Z height” may be used throughout to describe adistance of the objective lens with respect to the sample, this termdoes not limit the disclosed embodiments to an objective lens positionedabove the sample, but should instead be understood to encompass anydistance that represents a distance between the objective lens and aplane of the sample, regardless of their orientations to each other.

FIG. 1 is a block diagram illustrating an example side viewconfiguration of a scanning system 11, according to an embodiment. Inthe illustrated embodiment, scanning system 11 comprises a sample 120(e.g., a tissue sample prepared on a glass microscope slide) that isplaced on a motorized stage (not shown), illuminated by an illuminationsystem (not shown), and moved in a scanning direction 65. An objectivelens 130 has an optical field of view (FOV) 250 that is trained onsample 120 and provides an optical path for light from the illuminationsystem that passes through the specimen on the slide, reflects off ofthe specimen on the slide, fluoresces from the specimen on the slide, orotherwise passes through objective lens 130.

FIG. 1 illustrates the relative positions between an imaging sensor 20and a focusing sensor 30 in space. The light travels on the optical paththrough objective lens 130 to a beam splitter 140 that allows some ofthe light to pass through lens 160 to imaging sensor 20. As illustratedin FIG. 1, the light may be bent by a mirror 150 (e.g., at 90°) betweenlens 160 and imaging sensor 20. Imaging sensor 20 may be, for example, aline charge-coupled device (CCD) or a line complementary metal-oxidesemiconductor (CMOS) device.

In addition, some of the light travels from beam splitter 140 throughlens 165 to focusing sensor 30. As illustrated in FIG. 1, this light maybe bent by beam splitter 140 (e.g., at 90°) between objective lens 130and lens 165. Focusing sensor 30 may also be, for example, a linecharge-coupled device (CCD) or line CMOS device.

In an embodiment, the light that travels to imaging sensor 20 and thelight that travels to focusing sensor 30 each represents the completeoptical field of view 250 from objective lens 130. Based on theconfiguration of the system, the scanning direction 65 of sample 120 islogically oriented with respect to imaging sensor 20 and focusing sensor30 such that the logical scanning direction 60 causes the optical fieldof view 250 of objective lens 130 to pass over the respective focusingsensor 30 and imaging sensor 20.

FIG. 2 is a block diagram illustrating an example configuration offocusing sensor 30 and imaging sensor 20 with respect to an opticalfield of view 250 having a circular illumination radius 240, accordingto an embodiment. In the illustrated embodiment, the positioning offocusing sensor 30 is shown with respect to imaging sensor 20 and thelogical scan direction 60. In this case, the scan direction 60 refers tothe direction in which the stage or specimen (e.g., a tissue sample) ismoving with respect to sensors 30 and 20 in space. As illustrated,imaging sensor 20 is centered within optical field of view 250 ofobjective lens 130, while focusing sensor 30 is offset from the centerof optical field of view 250 of objective lens 130. The direction inwhich focusing sensor 30 is offset from the center of optical field ofview 250 of the objective lens 130 is the opposite of the logicalscanning direction 60. This placement logically orients focusing sensor30 in front of imaging sensor 20, such that, as a specimen on a slide isscanned, focusing sensor 30 senses the image data before, at the sametime that, or after the imaging sensor 20 senses that same image data.Thus, a given portion (e.g., line) of sample 120 will reach focusingsensor 30 first, and subsequently reach imaging sensor 20 second.

When imaging sensor 20 and focusing sensor 30 are projected onto a sameplane using, for example a beam-splitter, focusing sensor 30 is withinthe illumination circle, which has a radius R, of the optical field ofview 250 at a location ahead of the primary imaging sensor 20 in termsof the logical scanning direction 60. Thus, when a view of a section ofa tissue sample passes focusing sensor 30, focus data can be capturedand the focus height for objective lens 130 can be calculated based onone or more predetermined algorithms, prior to, at the same time as, orafter the time of the view of the same section of tissue sample passingimaging sensor 20. The focus data and the calculated focus height forobjective lens 130 can be used to control (e.g., by a controller) theheight of objective lens 130 from sample 120 before the view of the samesection of the tissue sample is sensed by imaging sensor 20 viaobjective lens 130. In this manner, imaging sensor 20 senses the view ofthe section of the tissue sample while objective lens 130 is at thecalculated focus height.

Circular illumination radius 240 preferably illuminates an optical fieldof view 250 covering both focusing sensor 30 and imaging sensor 20.Radius 240 is a function of the field of view on sample 120 and theoptical magnification of the focusing optical path M_(focusing). Thefunction can be expressed as:2R=FOV*M _(focusing)

For example, for M_(focusing)=20 and FOV=1.25 mm (e.g., Leica PlanApo20× objective), R=12.5 mm. Imaging sensor 20 is projected in the middleof the optical field of view 250 for best image quality, while focusingsensor 30 is offset with respect to the center of optical field of view250 by a distance h from imaging sensor 20. There is a relationshipamong the distance h, the radius R, and the length L of focusing sensor30, such that:h≤square root(R ²−(L/2)²)

For example, for a sensor length=20.48 mm and R=12.5 mm, h≤7.2 mm. Itshould be understood that, when h>0, any given region of sample 120 issensed by focusing sensor 30 first and imaging sensor 20 second,whereas, when h<0, the given region of sample 120 is sensed by imagingsensor 20 first and focusing sensor 30 second. If h=0, the given regionof sample 120 is sensed by imaging sensor 20 and focusing sensor 30simultaneously as the stage moves along slide scan direction 65. In anembodiment, the average of multiple line images of the same region ofsample 120 may be used as the line image for that region.

The available time t for focusing sensor 30 to capture multiple cameralines, for focus height calculation and for moving objective lens 130 tothe right focus height, is a function of the distance h between focusingsensor 30 and imaging sensor 20, magnification M_(focusing), and scanspeed v:v*t=h/M _(focusing)

For example, for a scan speed of 4.6 mm/s, the maximum time available isabout 78.3 ms for M_(focusing)=20 and h=7.2 mm. The maximum number ofcamera lines captured by focusing sensor 30, available for the focuscalculation is:N=t*κ, where κ is the line rate of the focusing sensor 30.

For example, for a camera line rate of 18.7 kHz, N_(max)=1,464 lines,where objective lens 130 stays at the same height. Otherwise, N<N_(max)to allow objective lens 130 to move to the next focus height.

At a high level, a sample 120 (e.g., tissue sample) is passed underobjective lens 130 in an X direction. A portion of sample 120 isilluminated to create an illuminated optical field of view 250 in the Zdirection of a portion of sample 120 (i.e., perpendicular to an X-Yplane of sample 120). The illuminated optical field of view 250 passesthrough objective lens 130 which is optically coupled to both focusingsensor 30 and imaging sensor 20, for example, using a beam splitter 140.Focusing sensor 30 and imaging sensor 20 are positioned, such thatfocusing sensor 30 receives a region or line of the optical field ofview 250 before, at the same time as, or after imaging sensor 20receives the same region or line. In other words, as focusing sensor 30is receiving a first line of image data, imaging sensor 20 issimultaneously receiving a second line of image data which waspreviously received by focusing sensor 30 and which is a distanceh/M_(focusing) on sample 120 from the first line of image data. It willtake a time period Δt for imaging sensor 20 to receive the first line ofimage data after focusing sensor 30 has received the first line of imagedata, where Δt represents the time that it takes sample 120 to move adistance h/M_(focusing) in the logical scan direction 60.

During the period Δt, a processor of scanning system 10 calculates anoptimal focus height in the Z direction for the first line of imagedata, and adjusts objective lens 130 to the calculated optimal focusdistance before, at the time that, or after imaging sensor 20 receivesthe first line of image data.

In an embodiment, focusing sensor 30 is separate from imaging sensor 20and is tilted at an angle θ with respect to a direction that isperpendicular to the optical imaging path. Thus, for each line of imagedata, focusing sensor 30 simultaneously receives pixels of image data ata plurality of Z height values. The processor may then determine thepixel(s) having the best focus within the line of image data (e.g.,having the highest contrast with respect to the other pixels within theline of image data). After the optimal Z height value is determined, theprocessor or other controller may move objective lens 130 in the Zdirection to the determined optimal Z height value before,simultaneously as, or after imaging sensor 20 receives the same line ofimage data.

As discussed above, focusing sensor 30 may be tilted within the opticalfield of view such that light from objective lens 130 is sensed byfocusing sensor 30 at a plurality of Z height values. FIG. 3A is a blockdiagram illustrating an example top view configuration of imaging sensor20 with respect to an imaging optical path 210, according to anembodiment. FIG. 3B is a block diagram illustrating an example top viewconfiguration of a tilted focusing sensor 30, with respect to a focusingoptical path 200, according to an embodiment. As can be seen in FIG. 3B,focusing sensor 30 is tilted at an angle θ with respect to a directionthat is perpendicular to focusing optical path 200. FIG. 3C is a blockdiagram illustrating an example of a sensor, in which half of the sensoris used to acquire a main image, and the other half of the sensor isused to acquire a focusing image.

Thus, an image projected on tilted focusing sensor 30 and acquired as aline of image data by tilted focusing sensor 30 will have variablesharpness or contrast. This line of image data will have its highestfocus (e.g., greatest sharpness or contrast) in a particular region orpixel location of tilted focusing sensor 30. Each region or pixellocation of tilted focusing sensor 30 may be directly mapped orotherwise correlated to a Z height of objective lens 130, such that theZ height of objective lens 130 may be determined from a particular pixellocation of tilted focusing sensor 30. Thus, once the pixel location ofhighest focus (e.g., highest contrast) is determined, the Z height ofobjective lens 130 providing the highest focus may be determined byidentifying the Z height of objective lens 130 that is mapped to thatpixel location of highest focus. Accordingly, a feedback loop may beconstructed. By this feedback loop, for a given region on sample 120,the position of objective lens 130 may be automatically controlled(e.g., by increasing or decreasing the height of objective lens 130) toalways correspond to the position on tilted focusing sensor 30 havingthe highest focus for that region, before, at the time that, or afterimaging sensor 20 senses the same region of sample 120, such that theregion of sample 120 being imaged by imaging sensor 20 is always at thebest available focus.

FIG. 4 is a block diagram illustrating an example tilted focusing sensor30, according to an embodiment. In the illustrated embodiment, tiltedfocusing sensor 30 comprises a plurality of sensor pixels 218 within arange of focusing (z) on a tissue sample (e.g., 20 μm). As illustratedin FIG. 4, tilted focusing sensor 30 may be positioned at a locationwhere the entire focusing range (z) in the Z direction is transferred byoptics to the entire array of sensor pixels 218 in tilted focusingsensor 30 in the Y direction. The location of each sensor pixel 218 isdirectly correlated or mapped to a Z height of objective lens 130. Asillustrated in FIG. 4, each dashed line, p₁, p₂, . . . p_(i) . . .p_(n), across projected focusing range (d) represents a different focusvalue and corresponds to a different focus height of objective lens 130.The p_(i) having the highest focus for a given region of a sample can beused by scanning system 11 to determine the optimal focus height ofobjective lens 130 for that region of sample 120.

The relationship between the projected focusing range (d) on tiltedfocusing sensor 30 and the focusing range (z) on sample 120 may beexpressed as: d=z*M_(focusing) ², where M_(focusing) is the opticalmagnification of the focusing path. For instance, if z=20 μm andM_(focusing)=20, then d=8 mm.

In order to cover the entire projected focusing range (d) by a tiltedfocusing sensor 30 that is a linear array sensor, the tilting angle θshould follow the relationship: sine, =d/L, where L is the length offocusing sensor 30. Using d=8 mm and L=20.48 mm, 0=23.0°. θ and L canvary as long as tilted focusing sensor 30 covers the entire focusingrange (z).

The focusing resolution, or the minimum step of focus height motion Δzalong the Z axis is a function of the sensor pixel size, e=minimum(ΔL).Derived from the above formulas: Δz=e*z/L. For instance, if e=10 μm,L=20.48 mm, and z=20 μm, then Δz=0.0097 μm<10 nm.

In an embodiment, a scan line (e.g., one-dimensional image data),acquired by tilted focusing sensor 30 from sample 120, is analyzed. Afigure of merit (FOM) (e.g., contrast of the data) may be defined. Thelocation (corresponding to a focus height value of objective lens 130)of a pixel 218 of the maximum FOM on the sensor array can be found. Inthis manner, the focus height of objective lens 130, corresponding tothe location of the pixel 218 of the maximum FOM, can be determined forthat scan line.

The relationship between the location L_(i) on tilted focusing sensor 30of a pixel i and the focus height Z_(i) of objective lens 130 may berepresented as follows: L_(i)=Z_(i)*M_(focusing) ²/sin θ

If the focus distance is determined by a mean from L₁ to L₂, accordingto the analysis of the data from tilted focusing sensor 30 discussedabove, the focus height of objective lens 130 needs to be moved from Z₁to Z₂ based on: Z₂=Z₁+(L₂−L₁)*sin θ/M_(focusing) ²

Although the field of view (FOV) in the Y axis of focusing sensor 30 andimaging sensor 20 can be different, the centers of both sensors arepreferably aligned to each other along the Y axis.

FIG. 5 is a time chart diagram illustrating an example interplay betweena focusing sensor 30 and an imaging sensor 20 during scanning, accordingto an embodiment. Specifically, the timing of a scan using an imagingsensor 20 and focusing sensor 30 is illustrated. At time t₀, the focusheight of objective lens 130 is at Z₀ on tissue section X₁, which is inthe field of view of focusing sensor 30. Focusing sensor 30 receivesfocusing data corresponding to the tissue section X₁. The focus heightZ₁ is determined to be the optimal focus height for tissue section X₁using the focusing data and, in some embodiments, associated focusingalgorithms. The optimal focus height is then fed to the Z positioner tomove objective lens 130 to the height Z₁, for example, using a controlloop. At t₁, tissue section X₁ is moved into the field of view ofimaging sensor 20. With the correct focus height, imaging sensor 20 willsense an optimally-focused image of tissue section X₁. At the same timet₁, focusing sensor 30 captures focusing data from tissue section X₂,and the focusing data will be used to determine the optimal focus heightZ₂ which in turn will be fed into the Z positioner prior to, at the timethat, or after tissue section X₂ passes into the field of view ofimaging sensor 20 at time t₂. Such a process can continue until theentire tissue sample is scanned.

In general at time t₀, tissue section X_(n+1) is in the field of view offocusing sensor 30, tissue section X_(n) is in the field of view ofimaging sensor 30, and objective lens 130 is at a focus height of Z_(n).Furthermore, prior to, at the time, or after t_(n+1), the optimal focusheight for tissue section X_(n+1) is determined and the focus height ofobjective lens 130 is adjusted to Z_(n+1). At time t₀, focusing sensor30 senses tissue section X₁ and determines the focus height as Z₁ fortissue section X₁; at time t₁, tissue section X₁ moves under imagingsensor 20 and objective lens 130 moves to focus height Z₁ while focusingsensor 30 senses tissue section X₂ and determines the focus height as Z₂for tissue section X₂; at time t_(n), tissue section X_(n) moves underimaging sensor 20 and objective lens 130 moves to focus height Z_(n)while focusing sensor 30 senses tissue section X_(n+1) and determinesthe focus height as Z_(n+1) for tissue section X_(n+1.) X_(n−1) andX_(n) do not necessarily represent consecutive or adjacent lines ofimage data, as long as a scan line is acquired by focusing sensor 30 andan optimal focus height for the scan line is determined and set priorto, at the same time as, or after the same scan line being acquired byimaging sensor 20. In other words, focusing sensor 30 and imaging sensor20 may be arranged such that one or more scan lines exist between thefield of view of focusing sensor 30 and the field of view of imagingsensor 20, i.e., that distance h between focusing sensor 30 and imagingsensor 20 comprises one or more scan lines of data. For instance, in thecase that the distance h comprises five scan lines, tissue section X₆would be in the field of view of focusing sensor 30 at the same timethat tissue section X₁ is in the field of view of imaging sensor 20. Inthis case, the focus height of objective lens 130 would be adjusted tothe calculated optimal focus height after the tissue section X₅ issensed by imaging sensor 20 but prior to, at the same time as, or afterthe tissue section X₆ being sensed by imaging sensor 20. Advantageously,the focus height of objective lens 130 may be smoothly controlledbetween tissue section X₁ and X₆ such that there are incremental changesin focus height between X₁ and X₆ that approximate a gradual slope ofthe tissue sample.

FIG. 6 illustrates a tilted focusing sensor 30 that utilizes one or morebeam splitters and one or more prism mirrors, according to anembodiment. The beam splitter(s) and mirror(s) are used to create aplurality of images of the same field of view on tilted focusing sensor30, with each of the plurality of images at a different focal distance,thereby enabling focusing sensor 30 to simultaneously sense multiplesimages of the same region of sample 120 at different foci (correspondingto different focus heights for objective lens 130). Focusing sensor 30may be a single large line sensor, or may comprise a plurality of linesensors (e.g., positioned in a row along a longitudinal axis).

Specifically, FIG. 6 illustrates a tilted focusing sensor 30 thatutilizes beam splitters 620A and 620B and prism mirror 630 to guide alight beam 605 (illustrated as separate red, blue, and green channels,although they do not need to be separated channels) through a pluralityof optical paths 610A-610C, each having different focal distances, ontoa single tilted line sensor 30. It should be understood that light beam605 conveys a field of view from objective lens 130. As illustrated, theoptical paths, in order from highest focal distance to lowest focaldistance, are 610A, 610B, and 610C. However, it should be understoodthat each optical path will reach tilted line sensor 30 at a range offocal distances, rather than at a single focal distance, due to the tiltof tilted line sensor 30. In other words, the image acquired by tiltedline sensor 30 on each optical path will comprise pixels acquired at afocal distance that increases from a first side of the image to asecond, opposite side of the image. In the illustrated example, lightbeam 605 enters beam splitter 620A and is split into an optical path610A, which proceeds to a first region of tilted focusing sensor 30 at afirst focal distance, and an optical path which proceeds to beamsplitter 620B. The optical path that proceeds to beam splitter 620B issplit into an optical path 610B, which proceeds to a second region oftilted focusing sensor 30 at a second focal distance, and an opticalpath 610C, which is reflected off of mirror 630 onto a third region oftilted focusing sensor 30 at a third focal distance. Each of the first,second, and third focal distances and first, second, and third regionsare different from each other. In this manner, a single tilted focusingsensor 30 simultaneously senses light beam 605 at a plurality ofdifferent focal distances (e.g., three in the illustrated example). Itshould be understood that fewer or more beam splitters 620 and/ormirrors 630 may be used to create fewer or more optical paths 610 withdifferent focal distances (e.g., two optical paths or four or moreoptical paths, each with a different focal distance with respect totilted focusing sensor 30).

With respect to the embodiment illustrated in FIG. 6, the best focus maybe determined and correlated to a height of objective lens 130, in thesame manner as described above. Redundant information from a pluralityof images at different focal distances may provide higher confidence toa focus result.

FIGS. 7A and 7B illustrate alternatives to a tilted focusing sensor.Specifically, FIGS. 7A and 7B illustrate a non-tilted focusing sensor 30that utilizes one or more beam splitters and one or more prism mirrorsto achieve the same results as a tilted focusing sensor, according to acouple of embodiments. The beam splitter(s) and mirror(s) are used tocreate a plurality of images of the same field of view on focusingsensor 30, with each of the plurality of images at a different focaldistance, thereby enabling focusing sensor 30 to simultaneously sensemultiple images of the same region of sample 120 at different foci(corresponding to different focus heights for objective lens 130).Focusing sensor 30 may be a single large line sensor, or may comprise aplurality of line sensors positioned in a row along a longitudinal axis.

FIG. 7A illustrates a non-tilted focusing sensor 30 that utilizes beamsplitters 620A and 620B and prism mirrors 630A and 630B to guide a lightbeam 605 (illustrated as separate red, blue, and green channels,although they do not need to be separated channels) through a pluralityof optical paths 610A-610C, each having different focal distances, ontoa single line sensor 30. It should be understood that light beam 605conveys a field of view from objective lens 130. As illustrated, theoptical paths, in order from highest focal distance to lowest focaldistance, are 610A, 610B, and 610C. In the illustrated example, lightbeam 605 enters beam splitter 620A and is split into an optical path610B, which is reflected off of mirror 630A and through glass block 640Aonto a first region of focusing sensor 30 at a first focal distance, andan optical path which proceeds to beam splitter 620B. The optical paththat proceeds to beam splitter 620B is split into an optical path 610Awhich passes onto a second region of focusing sensor 30 (e.g., adjacentto the first region of focusing sensor 30) at a second focal distance,and an optical path 610C, which is reflected off of mirror 630B andthrough glass block 640B onto a third region of focusing sensor 30(e.g., adjacent to the second region of focusing sensor 30) at a thirdfocal distance. Each of the first, second, and third focal distances andfirst, second, and third regions are different from each other. In thismanner, focusing sensor 30 simultaneously senses light beam 605 at aplurality of different focal distances (e.g., three in the illustratedexample). It should be understood that fewer or more beam splitters 620,mirrors 630, glass blocks 640, and/or regions of focusing sensor 30 maybe used to create fewer or more optical paths 610 with different focaldistances (e.g., two optical paths or four or more optical paths, eachwith a different focal distance with respect to focusing sensor 30).

FIG. 7B illustrates a non-tilted focusing sensor 30 that utilizes beamsplitters 620A and 620B and prism mirrors 630A and 630B to guide a lightbeam 605 (illustrated as separate red, blue, and green channels,although they do not need to be separated channels) through a pluralityof optical paths 610A-610C, each having different focal distances, ontorespective ones of a plurality of line sensors 30A-30C. As illustrated,the optical paths, in order from highest focal distance to lowest focaldistance, are 610A, 610B, and 610C. In the illustrated example, lightbeam 605 enters beam splitter 620A and is split into an optical path610B, which is reflected off of mirror 630A and through glass block 640Aonto a first region of focusing sensor 30 at a first focal distance, andan optical path which proceeds to beam splitter 620B. The optical paththat proceeds to beam splitter 620B is split into an optical path 610Awhich passes onto a second region of focusing sensor 30 at a secondfocal distance, and an optical path 610C, which is reflected off ofmirror 630B and through glass block 640B onto a third region of focusingsensor 30 at a third focal distance. Each of the first, second, andthird focal distances and the first, second, and third regions offocusing sensor 30 are different from each other. In this manner,focusing sensor 30 simultaneously senses light beam 605 at a pluralityof different respective focal distances (e.g., three in the illustratedexample). It should be understood that fewer or more beam splitters 620,mirrors 630, glass blocks 640, and/or regions of focusing sensor 30 maybe used to create fewer or more optical paths 610 with different focaldistances (e.g., two optical paths or four or more optical paths, eachwith a different focal distance with respect to a different focusingsensor 30).

In the embodiments illustrated in FIGS. 6, 7A, and 7B, the beamsplitters and mirrors are positioned after the imaging lens in theoptical path. Alternatively, tube lenses may be positioned after thebeam splitting optics. In this alternative embodiment, the locations ofindividual images with the same field of view are defined by the focallengths and positions of the lenses.

FIG. 7C illustrates an alternative non-tilted focusing sensor 30 inwhich the beam splitting optics are positioned before tube lenses,according to an embodiment. Specifically, non-tilted focusing sensor 30utilizes beam splitters 620A, 620B, and 620C and prism mirror 630 toguide a light beam 605 through a plurality of optical paths 610A-610D,each having different focal distances, onto a single line sensor 30. Asillustrated, the optical paths, in order from highest focal distance tolowest focal distance, are 610A, 610B, 610C, and 610D. In theillustrated example, light beam 605 enters beam splitter 620A and issplit into an optical path 610A, which is focused by lens 650A onto afirst region of focusing sensor 30 at a first focal distance, and anoptical path which proceeds to beam splitter 620B. The optical path thatproceeds to beam splitter 620B is split into an optical path 610B, whichis focused by lens 650B onto a second region of focusing sensor 30 at asecond focal distance, and an optical path which proceeds to beamsplitter 620C. The optical path that proceeds to beam splitter 620B issplit into an optical path 610C, which is focused by lens 650C onto athird region of focusing sensor 30 at a third focal distance, and anoptical path 610C, which is reflected off of mirror 630 and focused bylens 650D onto a fourth region of tilted focusing sensor 30 at a fourthfocal distance. Each of the first, second, third, and fourth focaldistances and the first, second, third, and fourth regions are differentfrom each other. In this manner, focusing sensor 30 (e.g., comprising asingle line sensor or a plurality of line sensors) simultaneously senseslight beam 605 at a plurality of different focal distances (e.g., fourin the illustrated example). It should be understood that fewer or morebeam splitters 620, mirrors 630, and/or regions of focusing sensor 30may be used to create fewer or more optical paths 610 with differentfocal distances (e.g., two optical paths, three optical paths, or fiveor more optical paths, each with a different focal distance with respectto focusing sensor 30).

In the embodiments described above, a given region of a sample 120 issimultaneously acquired by different regions of a focusing sensor 30 ata plurality of different focal distances, producing a plurality ofimages at different focal distances. An algorithm can then be applied tothis plurality of images to determine a best focal distance, which canbe correlated to a focus height of objective lens 130 along the Z axis.

By aligning the optics (e.g., as discussed above), the plurality ofimages acquired by different regions of focusing sensor 30 can correlateor map to various focus spots from a focus buffer. The focus buffer maycontain contrast measures, for the focus points, calculated from imagedata that has been continuously acquired while objective lens 130 movesalong the Z axis (i.e., as the focus height of objective lens 130changes). For instance, a measure of contrast (e.g., averaged contrast)for each focus height represented by the plurality of images may beplotted, as illustrated in an example by the points in FIG. 8. The bestfocus (i.e., the peak of contrast measures in the focus buffer) can bedetermined by using a peak-finding algorithm (e.g., fitting,hill-climbing, etc.) to identify the peak of a curve that best fits thepoints, as illustrated in an example by the curve in FIG. 8. The peak ofthe curve represents the best contrast measure, and maps to a particularfocus height that provides the best focus.

FIG. 9A illustrates the focal relationship between a tilted focusingsensor 30 and imaging sensor 20, according to an embodiment.Specifically, in an embodiment, a point P of tilted focusing sensor 30is parfocal with imaging sensor 20. Thus, when sensing a region of asample 120 using tilted focusing sensor 30, the appropriate focus heightof objective lens 130 from sample 120 may be determined as the focusheight of objective lens 130 that positions the pixel(s) having the bestfocus at point P of focusing sensor 30. This determined focus height iswhat may then be used for objective lens 130 when sensing the sameregion using imaging sensor 20.

FIGS. 9B-9D illustrate the focus functions for tilted focusing sensor 30and imaging sensor 20. The focus function may be a function of contrastwithin the images sensed by tilted focusing sensor 30 and imaging sensor20. For example, C_(I) represents the contrast function for imagingsensor 20, and C_(T) represents the contrast function for tiltedfocusing sensor 30. Thus, C_(I)(x) returns a contrast measure for animage pixel at position x along the array of imaging sensor 20, andC_(T)(x) returns a contrast measure for an image pixel at position xalong the array of tilted focusing sensor 30. In both instances, thecontrast measure may be a root mean square of contrast values at x.C_(D) represents the difference between C_(T) and C_(I) (e.g.,C_(T)−C_(I)). Thus, C_(D)(x) represents the difference between C_(T) andC_(I) at position x along the arrays of imaging sensor 20 and tiltedfocusing sensor 30 (e.g., C_(T)(x)−C_(I)(x)). C_(D)(x) removestissue-dependent spatial variations. A ratio between the contrastfunctions of the two images can be used as well to removetissue-dependent spatial variations (e.g., C_(T)(x)/C_(I)(x)). Inaddition, a threshold can be defined to remove influences frombackground noise.

FIG. 9B illustrates the contrast function C_(I) ² which representscontrast measures as a function of a position on imaging sensor 20.Similarly, FIG. 9C illustrates the contrast function C_(T) ² whichrepresents contrast measures as a function of a position on tiltedfocusing sensor 30. FIG. 9D illustrates a ratio of the contrast functionfor tilted focusing sensor 30 to the contrast function for imagingsensor 20 (i.e., C_(T) ²/C_(I) ²).

When tilted focusing sensor 30 and imaging sensor 20 both sense the sameregion of sample 120, the best focus will be at a position x on bothsensors 30 and 20 at which the ratio of C_(T) to C_(I) (e.g.,C_(T)/C_(I)) is 1.0. A predetermined point P on tilted focusing sensor30 is parfocal with imaging sensor 20. This point P may be determinedduring system calibration.

In an embodiment, a figure-of-merit (FOM) function may be used todetermine the best focus for a region of sample 120 based on dataacquired by tilted focusing sensor 30. Specifically, a peak of the C_(T)function may be determined. This C_(T) peak will correspond to aposition x on tilted focusing sensor 30 and is correlated to a focusheight within the Z range of objective lens 130. Thus, when the C_(T)peak is away from the parfocal point P on tilted focusing sensor 30(i.e., C_(T)(P) does not represent the peak value), a command may beinitiated to move objective lens 130 in real time along an axis that isorthogonal to sample 120 (i.e., Z axis) until the peak of C_(T) is at P(i.e., until C_(T)(P) is the peak value for C_(T)). In other words,de-focus is characterized by the shift of the peak value of C_(T) awayfrom parfocal point P on tilted focusing sensor 30, and auto-focusingcan be achieved via a feedback loop that moves objective lens 130 alongthe Z axis until the peak value of C_(T) is at parfocal point P ontilted focusing sensor 30.

In an embodiment, an image of a field of view acquired by imaging sensor20 is compared to an image of the same field of view acquired byfocusing sensor 30 (e.g., tilted focusing sensor 30) using their ratio,difference, or other calculation.

In an embodiment, focusing sensor 30 may comprise a single line sensoror dual line sensors designed to acquire two images of the same field ofview that are reversed in terms of the focal distances represented bythe pixels of the images. For example, a first one of the two images haspixels representing the lowest focal distance at a first side (e.g.,left side) of the captured field of view and pixels representing thehighest focal distance at a second side of the captured field of viewthat is opposite the first side (e.g., right side), whereas the secondone of the two images has pixels representing the highest focal distanceat the first side (e.g., left side) of the captured field of view andpixels representing the lowest focal distance at the second side (e.g.,right side) of the captured field of view. If the highest and lowestfocal distances are the same for both images, then a line of pixels inthe center of each image will be parfocal between the two images, andthe corresponding lines of pixels emanating from the center to the sidesedges (e.g., left and right edges) of the captured field of view of eachimage will also be parfocal between the two images but in oppositedirections. For example, using left and right to arbitrarily define theedges of the images, for all distances D, a vertical line of pixels inthe first image that is a distance D from the center to the left edge ofthe field of view represented in the first image will be parfocal with avertical line of pixels in the second image that is a distance D fromthe center to the right edge of the field of view in the second image.It should be understood that, if the field of view is inverted ormirrored between the first and second images, then both images will havetheir highest and lowest focal distances on the same sides of the image,but on opposite sides of the field of view represented by the image.

FIG. 10 illustrates optical components for forming two images withreversed focal distances using two focusing sensors 30A and 30B,according to an embodiment. The tilt of focusing sensor 30A is reversedwith respect to the tilt of focusing sensor 30B around the logical Zaxis (i.e., the focus axis). It should be understood that the logical Zaxis in FIG. 10 is not necessarily the same as the physical Z axis ofobjective lens 130, since the light may be bent (e.g., orthogonally by abeam splitter or prism mirror) after it passes through objective lens130. In this embodiment, optical paths 610A and 610B provide the sameoptical field of view to focusing sensors 30A and 30B, but sincefocusing sensors 30A and 30B are reversed in terms of their tilt, thefocal distances of pixels in the two images are reversed. This isillustrated by the sets of three arrows labeled Z₁, Z₂, and Z₃, whicheach represent a different sample focal height. Thus, Z₁ ^(a) and Z₁^(b) both represent a first focal height, Z₂ ^(a) and Z₂ ^(b) bothrepresent a second focal height, and Z₁ ^(a) and Z₃ ^(b) both representa third focal height, where each of the first, second, and third focalheights are different from each other.

FIGS. 11A and 11B illustrate optical components for forming two mirrorimages on a tilted focusing sensor 30, according to two differentembodiments. In the illustrated embodiments, one or more opticalcomponents may be used to form the field of view on a first region oftilted focusing sensor 30 and a reversed field of view on a secondregion of tilted focusing sensor 30. It should be understood that tiltedfocusing sensor 30 may be a single line sensor or a plurality ofadjacent line sensors.

FIG. 11A illustrates optical components for forming two mirror images ona tilted focusing sensor 30, according to a first embodiment. Asillustrated, a light beam 605 enters beam splitter 620 and is split intoan optical path 610A, which is reflected off of mirror 630 onto a firstregion 30A of focusing sensor 30 such that a first image is acquiredfrom first region 30A of focusing sensor 30, and an optical path 610B,which passes through dove prism 660. Dove prism 660 reverses light beam605 so that a mirror image is formed on a second region 30B of focusingsensor 30 such that a second image is acquired from second region 30B offocusing sensor 30. In other words, optical path 610A provides the fieldof view to first region 30A of focusing sensor 30 and a mirrored fieldof view to second region 30B of focusing sensor 30. Thus, the secondimage is a mirror image of the first image around the logical Z axis.Since the angle of tilt (θ) is the same in both first region 30A andsecond region 30B of tilted focusing sensor 30, the fields of viewdepicted in the first and second images are reversed in terms of thedirection of the focal distances (e.g., from highest to lowest) at whichthey were acquired.

FIG. 11B illustrates optical components for forming two mirror images ona tilted focusing sensor 30, according to a second embodiment. Asillustrated, a light beam 605 enters beam splitter 620 and is split intoan optical path 610A, which is reflected off of mirror 630A (e.g., aflat plate) to a first region 30A of tilted focusing sensor 30, and anoptical path 610B. Optical path 610B reflects off of two surfaces ofmirror 630B back into beam splitter 620, where it is reflected onto asecond region 30B of tilted focusing sensor 30. Light beam 605 travelingon optical path 610B is reversed such that it produces an image onsecond region 30B of tilted focusing sensor 30 that is the mirror imageof the image formed in optical path 610A on first region 30A of tiltedfocusing sensor 30. Since the angle of tilt (θ) is the same in bothfirst region 30A and the second region 30B of tilted focusing sensor 30,the fields of view depicted in the first and second images are reversedin terms of the direction (e.g., from highest to lowest) of the focaldistances at which they were acquired.

FIG. 12A illustrates the directionality of the focal distances for thetwo images acquired by regions 30A and 30B of focusing sensor 30 in theembodiments illustrated in FIGS. 10, 11A, and 11B, according to anembodiment. A comparison of these two images using a difference, aratio, or another calculation may provide the amount of movement anddirection of movement required to place objective lens 130 at a focusheight on the Z axis that achieves the best focus for focusing sensor30. In an embodiment, the center or a parfocal point of each of theregion(s) of focusing sensor 30 (i.e., each region corresponding to itsown separate optical path 610) is parfocal with each other, as well aswith imaging sensor 20. Thus, determining a best focus for a givenregion of sample 120 comprises identifying a focus height for objectivelens 130, such that the best foci for the two images acquired byfocusing sensor 30 are at the center or a parfocal point of therespective regions of focusing sensor 30 that acquired the images. Whenthe best foci for the regions of focusing sensor 30 are centered orparfocal in this manner, the focus height of objective lens 130 thatcorresponds to the centers or parfocal points of both regions is alsothe focus height at which imaging sensor 30 (which is parfocal with thecenters of both regions of focusing sensor 30 or with a parfocal point,e.g., determined during system calibration) is at the best focus for thegiven region of sample 120.

FIG. 12B illustrates the focus functions for two reversed imagesacquired by regions 30A and 30B of focusing sensor 30. In embodiments inwhich a mirrored image is acquired (e.g., by region 30B in FIGS. 11A and11B), the mirrored image is inverted by software or other means prior tooperations performed with respect to the two reversed images. Thisinversion of the mirrored image results in the two images no longerbeing mirror images of each other in terms of content. In other words,the two images represent the same field of view in the same orientation.However, even though the orientation of the field of view represented bythe images are the same, the directions of their focal distances arereversed. For example, after this inversion process, the content on oneside of a first one of the images will have been acquired at focaldistance Z₁, while the same content on the same side of the second oneof the images will have been acquired at focal distance Z₃, and thecontent on the other side of the first image will have been acquired atfocal distance Z₃, while the same content on the same side of the secondimage will have been acquired at Z₁. The centers of the images will bothhave been acquired at Z₂.

The focus function may be a function of contrast within the reversedimages. The functions may return a contrast measure for a given positionx along each region of focusing sensor 30 (e.g., regions 30A and 30B)that acquires one of the reversed images (e.g., a root mean square ofcontrast values at a position x). For example, C^(b) represents thecontrast measures for region 30B of focusing sensor 30 which acquiredthe reversed image, and C^(a) represents the contrast measures forregion 30A of focusing sensor 30 which acquired the non-reversed image.C₂ ^(a) and C₂ ^(b) represent the contrast measures for the middleportions of the reversed images, C₁ ^(a) and C₁ ^(b) represent thecontrast measures for the corresponding portions of one side of thereversed images, and C₃ ^(a) and C_(a) ^(b) represent the contrastmeasures for the corresponding portions of the other side of thereversed images.

If a ratio algorithm is used, C₂ ^(a)/C₂ ^(b) will be close to 1.0across the entire field of view of focusing sensor 30 when the best focifor both images are centered in their corresponding regions (e.g.,regions 30A and 30B) of focusing sensor 30. When the minimum of C₁^(a)/C₁ ^(b) (i.e., C₁ ^(a)/C₁ ^(b)<1.0) is on the left side of parfocalpoint P, a command may be sent in a feedback loop to move objective lens130 along the Z axis in a direction such that the minimum of C₁ ^(a)/C₁^(b) moves towards parfocal point P. When the maximum of C₃ ^(a)/C₃ ^(b)(i.e., C₃ ^(a)/C₃ ^(b)>1.0) is on the left side of parfocal point P, acommand may be sent in a feedback loop to move objective lens 130 alongthe Z axis in a direction such that the maximum of C₃ ^(a)/C₃ ^(b) movestowards parfocal point P. The same algorithm may be applied to the otherhalf of the ratio data centered to parfocal point P (i.e., theright-hand side of the curve). The second set of data can be used incases where half of the field of view contains no tissue or non-usefuldata, or simply for redundancy to increase a success rate.

In any of the embodiments described herein as using multiple regions ofa focusing sensor 30 (e.g., the embodiments illustrated FIGS. 6-7C, 11A,and 11B), focusing sensor 30 may be a single focusing sensor comprisingthe multiple regions, or a plurality of focusing sensors each consistingof one of the multiple regions. Furthermore, in embodiments in which aplurality of focusing sensors are used as the regions of focusing sensor30, each of the plurality of focusing sensors may be arranged in thesame plane as each other, or in different planes from each other,depending on the particular design.

FIG. 13 illustrates a method for real-time focusing, according to anembodiment. Initially, a calibration step 1302 may be performed.Calibration step 1302 may comprise locating a parfocal point P (e.g.,parfocal with imaging sensor 20) on a tilted focusing sensor 30 (inembodiments which utilize a tilted focusing sensor), determining anillumination profile for images from imaging sensor 20, and/ordetermining an illumination profile for images from focusing sensor 30.It should be understood that calibration step 1302 may be performed onlyonce for a particular system 11, or periodically for the system 11 ifrecalibration is needed or desired.

The real-time focusing process may begin in step 1304, in which one ormore, and preferably a plurality of three or more, focus points areacquired using a focus-buffer method. Each focus point may comprise anX, Y, and Z position, where the X and Y positions represent a positionin a plane of sample 120, and the Z position represents a focus heightof objective lens 130. In an embodiment, each focus point is obtained bypositioning objective lens 130 over an X-Y position on sample 120,sweeping objective lens 130 from one end of its height range to theother end of its height range to determine the focus height providingthe best focus (e.g., peak of a contrast function) at the X-Y position.

In step 1306, a reference plane is created using the focus pointsobtained in step 1304. It should be understood that a reference planecan be created from as few as three focus points. When there are morethan three focus points, focus points that are outliers with respect toa flat reference plane may be discarded. Otherwise, all focus points maybe used to fit a reference plane. Alternatively, instead of a referenceplane, a focal surface may be created from any plurality of focuspoints. Different embodiments for creating a reference plane or focalsurface are described in U.S. patent application Ser. No. 09/563,437,filed on May 3, 2000 and issued as U.S. Pat. No. 6,711,283 on Mar. 23,2004, and U.S. patent application Ser. No. 10/827,207, filed on Apr. 16,2004 and issued as U.S. Pat. No. 7,518,652 on Apr. 14, 2009, theentireties of both of which are hereby incorporated herein by reference.

In step 1308, objective lens 130 is moved to a Z position defined by thereference plane as a function of the X-Y position to be scanned.

In step 1310, a focusing image is acquired from focusing sensor 30.Similarly, in step 1320, a main image is acquired from imaging sensor20.

In step 1312, the illumination in the focusing image acquired in step1310 is corrected using any well-known illumination-correctiontechnique. Similarly, in step 1322, the illumination in the main imageacquired in step 1320 is corrected using any well-knownillumination-correction techniques. The illumination correction for thefocusing image may be based on the illumination profile for focusingsensor 30 that was determined in calibration step 1302, and theillumination correction for the main image may be based on theillumination profile for imaging sensor 20 that was determined incalibration step 1302.

In step 1314, an absolute gradient of the illumination-correctedfocusing image is calculated. Similarly, in step 1324, an absolutegradient of the illumination-corrected main image is calculated.

In step 1316, the rows in the focusing image gradient calculated in step1314 are averaged. Similarly, in step 1326, the rows in the main imagegradient calculated in step 1324 are averaged.

In step 1318, a low-pass filter is applied to the focusing imagegradient. Similarly, in step 1328, a low-pass filter is applied to themain image gradient.

In step 1330, it is determined whether or not the background area (i.e.,the area of the image without tissue) in the main image is less than thetissue area (i.e., the area of the image with tissue) in the main image.If the background area is greater than the tissue area in the main image(i.e., “No” in step 1330), the process may return to step 1308.Otherwise, if the background area is less than the tissue area in themain image (i.e., “Yes” in step 1330), the process may proceed to step1332.

In step 1332, ratio(s) are calculated between the focusing imagegradient and the main image gradient. For example, the focusing imagegradient may be divided by the main image gradient.

In step 1334, a peak is fit to the ratio(s) calculated in step 1332 withminimal error. For example, a best-fit curve may be found for theratio(s).

In step 1336, the peak of the fitting in step 1334 is determined. Forexample, in an embodiment in which a best-fit curve is found for theratio(s) in step 1334, the peak of the best-fit curve may be identifiedin step 1336.

In step 1338, if the peak identified in step 1336 is not at the parfocalpoint P, objective lens 130 is moved until the peak is at the parfocalpoint P, for example, using a feedback loop as described elsewhereherein.

In step 1340, it is determined whether or not the scan is complete. Ifthe scan is not complete (i.e., “No” in step 1340), the process returnsto steps 1310 and 1320. Otherwise, if the scan is complete (i.e., “Yes”in step 1340), the process ends.

FIGS. 14A and 14B are block diagrams illustrating example microscopeslide scanners, according to an embodiment, and FIG. 14C is a blockdiagram illustrating example linear sensor arrays, according to anembodiment. These three figures will be described in more detail below.However, they will first be described in combination to provide anoverview. It should be noted that the following description is just anexample of a slide scanner device and that alternative slide scannerdevices can also be employed. FIGS. 14A and 14B illustrate examplemicroscope slide scanners that can be used in conjunction with thedisclosed sensor arrangement. FIG. 14C illustrates example linearsensors, which can be used in any combination as the disclosed sensors(imaging sensor 20 or focusing sensor 30).

For example, imaging sensor 20 and focusing sensor 30 may be arranged,as discussed above, using line scan camera 18 as primary imaging sensor20. In one embodiment, line scan camera 18 may include both focusingsensor 30 and imaging sensor 20. Imaging sensor 20 and focusing sensor30 can receive image information from a sample 120 through themicroscope objective lens 130 and/or the focusing optics 34 and 290.Focusing optics 290 for focusing sensor 30 may comprise the various beamsplitters 620, mirrors 630, and glass blocks 640 illustrated in FIGS.6-8. Imaging sensor 20 and focusing sensor 30 can provide informationto, and/or receive information from, data processor 21. Data processor21 is communicatively connected to memory 36 and data storage 38. Dataprocessor 21 may further be communicatively connected to acommunications port, which may be connected by at least one network 42to one or more computers 44, which may in turn be connected to displaymonitor(s) 46.

Data processor 21 may also be communicatively connected to and provideinstructions to a stage controller 22, which controls a motorized stage14 of slide scanner 11. Motorized stage 14 supports sample 120 and movesin one or more directions in the X-Y plane. In one embodiment, motorizedstage 14 may also move along the Z axis. Data processor 21 may also becommunicatively connected to and provide instructions to a motorizedcontroller 26, which controls a motorized positioner 24 (e.g., a piezopositioner). Motorized positioner 24 is configured to move objectivelens 130 in the Z axis. Slide scanner 11 also comprises a light source31 and/or illumination optics 32 to illuminate sample 120, either fromabove or below.

FIG. 14A is a block diagram of an embodiment of an optical microscopysystem 10, according to an embodiment. The heart of system 10 is amicroscope slide scanner 11 that serves to scan and digitize a specimenor sample 120. Sample 120 can be anything that may be interrogated byoptical microscopy. For instance, sample 120 may comprise a microscopeslide or other sample type that may be interrogated by opticalmicroscopy. A microscope slide is frequently used as a viewing substratefor specimens that include tissues and cells, chromosomes, DNA, protein,blood, bone marrow, urine, bacteria, beads, biopsy materials, or anyother type of biological material or substance that is either dead oralive, stained or unstained, labeled or unlabeled. Sample 120 may alsobe an array of any type of DNA or DNA-related material such as cDNA orRNA or protein that is deposited on any type of slide or othersubstrate, including any and all samples commonly known as microarrays.Sample 120 may be a microtiter plate, for example a 96-well plate. Otherexamples of sample 120 include integrated circuit boards,electrophoresis records, petri dishes, film, semiconductor materials,forensic materials, or machined parts.

Scanner 11 includes a motorized stage 14, a microscope objective lens130, a line scan camera 18, and a data processor 21. Sample 120 ispositioned on motorized stage 14 for scanning. Motorized stage 14 isconnected to a stage controller 22 which is connected in turn to dataprocessor 21. Data processor 21 determines the position of sample 120 onmotorized stage 14 via stage controller 22. In an embodiment, motorizedstage 14 moves sample 120 in at least the two axes (x/y) that are in theplane of sample 120. Fine movements of sample 120 along the opticalz-axis may also be necessary for certain applications of scanner 11, forexample, for focus control. Z-axis movement may be accomplished with apiezo positioner 24, such as the PIFOC from Polytec PI or the MIPOS 3from Piezosystem Jena. Piezo positioner 24 is attached directly tomicroscope objective 130 and is connected to and directed by dataprocessor 21 via piezo controller 26. A means of providing a coarsefocus adjustment may also be needed and can be provided by Z-axismovement as part of motorized stage 14 or a manual rack-and-pinioncoarse focus adjustment (not shown).

In one embodiment, motorized stage 14 includes a high-precisionpositioning table with ball bearing linear ways to provide smooth motionand excellent straight line and flatness accuracy. For example,motorized stage 14 could include two Daedal model 106004 tables stackedone on top of the other. Other types of motorized stages 14 are alsosuitable for scanner 11, including stacked single-axis stages based onways other than ball bearings, single- or multiple-axis positioningstages that are open in the center and are particularly suitable fortrans-illumination from below the sample, or larger stages that cansupport a plurality of samples. In one embodiment, motorized stage 14includes two stacked single-axis positioning tables, each coupled to twomillimeter lead-screws and Nema-23 stepping motors. At the maximum leadscrew speed of twenty-five revolutions per second, the maximum speed ofsample 120 on the motorized stage 14 is fifty millimeters per second.Selection of a lead screw with larger diameter, for example fivemillimeters, can increase the maximum speed to more than 100 millimetersper second. Motorized stage 14 can be equipped with mechanical oroptical position encoders which has the disadvantage of addingsignificant expense to the system. Consequently, such an embodiment doesnot include position encoders. However, if one were to use servo motorsin place of stepping motors, then one would have to use positionfeedback for proper control.

Position commands from data processor 21 are converted to motor currentor voltage commands in stage controller 22. In one embodiment, stagecontroller 22 includes a 2-axis servo/stepper motor controller(Compumotor 6K2) and two 4-amp microstepping drives (Compumotor OEMZL4).Microstepping provides a means for commanding the stepper motor in muchsmaller increments than the relatively large single 1.8 degree motorstep. For example, at a microstep of 100, sample 120 can be commanded tomove at steps as small as 0.1 micrometer. In an embodiment, a microstepof 25,000 is used. Smaller step sizes are also possible. It should beunderstood that the optimum selection of motorized stage 14 and stagecontroller 22 depends on many factors, including the nature of sample120, the desired time for sample digitization, and the desiredresolution of the resulting digital image of sample 120.

Microscope objective lens 130 can be any microscope objective lenscommonly available. One of ordinary skill in the art will recognize thatthe choice of which objective lens to use will depend on the particularcircumstances. In an embodiment, microscope objective lens 130 is of theinfinity-corrected type.

Sample 120 is illuminated by an illumination system 28 that includes alight source 31 and illumination optics 32. In an embodiment, lightsource 31 includes a variable intensity halogen light source with aconcave reflective mirror to maximize light output and a KG-1 filter tosuppress heat. However, light source 31 could also be any other type ofarc-lamp, laser, light emitting diode (“LED”), or other source of light.In an embodiment, illumination optics 32 include a standard Köhlerillumination system with two conjugate planes that are orthogonal to theoptical axis. Illumination optics 32 are representative of thebright-field illumination optics that can be found on mostcommercially-available compound microscopes sold by companies such asLeica, Carl Zeiss, Nikon, or Olympus. One set of conjugate planesincludes (i) a field iris aperture illuminated by light source 31, (ii)the object plane that is defined by the focal plane of sample 120, and(iii) the plane containing the light-responsive elements of line scancamera 18. A second conjugate plane includes (i) the filament of thebulb that is part of light source 31, (ii) the aperture of a condenseriris that sits immediately before the condenser optics that are part ofillumination optics 32, and (iii) the back focal plane of the microscopeobjective lens 130. In an embodiment, sample 120 is illuminated andimaged in transmission mode, with line scan camera 18 sensing opticalenergy that is transmitted by sample 120, or conversely, optical energythat is absorbed by sample 120.

Scanner 11 is equally suitable for detecting optical energy that isreflected from sample 120, in which case light source 31, illuminationoptics 32, and microscope objective lens 130 must be selected based oncompatibility with reflection imaging. A possible embodiment maytherefore include illumination through a fiber optic bundle that ispositioned above sample 120. Other possibilities include excitation thatis spectrally conditioned by a monochromator. If microscope objectivelens 130 is selected to be compatible with phase-contrast microscopy,then the incorporation of at least one phase stop in the condenseroptics that are part of illumination optics 32 will enable scanner 11 tobe used for phase contrast microscopy. To one of ordinary skill in theart, the modifications required for other types of microscopy such asdifferential interference contrast and confocal microscopy should bereadily apparent. Overall, scanner 11 is suitable, with appropriate butwell-known modifications, for the interrogation of microscopic samplesin any known mode of optical microscopy.

Between microscope objective lens 130 and line scan camera 18 aresituated line scan camera focusing optics 34 that focus the opticalsignal captured by microscope objective lens 130 onto thelight-responsive elements of line scan camera 18 (e.g., imaging sensor20). In a modern infinity-corrected microscope, the focusing opticsbetween the microscope objective lens and the eyepiece optics, orbetween the microscope objective lens and an external imaging port,comprise an optical element known as a tube lens that is part of amicroscope's observation tube. Many times the tube lens consists ofmultiple optical elements to prevent the introduction of coma orastigmatism. One of the motivations for the relatively recent changefrom traditional finite tube length optics to infinity corrected opticswas to increase the physical space in which the optical energy fromsample 120 is parallel, meaning that the focal point of this opticalenergy is at infinity. In this case, accessory elements like dichroicmirrors or filters can be inserted into the infinity space withoutchanging the optical path magnification or introducing undesirableoptical artifacts.

Infinity-corrected microscope objective lenses are typically inscribedwith an infinity mark. The magnification of an infinity correctedmicroscope objective lens is given by the quotient of the focal lengthof the tube lens divided by the focal length of the objective lens. Forexample, a tube lens with a focal length of 180 millimeters will resultin 20× magnification if an objective lens with 9 millimeter focal lengthis used. One of the reasons that the objective lenses manufactured bydifferent microscope manufacturers are not compatible is because of alack of standardization in the tube lens focal length. For example, a20× objective lens from Olympus, a company that uses a 180 millimetertube lens focal length, will not provide a 20× magnification on a Nikonmicroscope that is based on a different tube length focal length of 200millimeters. Instead, the effective magnification of such an Olympusobjective lens engraved with 20× and having a 9 millimeter focal lengthwill be 22.2×, obtained by dividing the 200 millimeter tube lens focallength by the 9 millimeter focal length of the objective lens. Changingthe tube lens on a conventional microscope is virtually impossiblewithout disassembling the microscope. The tube lens is part of acritical fixed element of the microscope. Another contributing factor tothe incompatibility between the objective lenses and microscopesmanufactured by different manufacturers is the design of the eyepieceoptics, the binoculars through which the specimen is observed. Whilemost of the optical corrections have been designed into the microscopeobjective lens, most microscope users remain convinced that there issome benefit in matching one manufacturers' binocular optics with thatsame manufacturers' microscope objective lenses to achieve the bestvisual image.

Line scan camera focusing optics 34 include a tube lens optic mountedinside of a mechanical tube. Since scanner 11, in an embodiment, lacksbinoculars or eyepieces for traditional visual observation, the problemsuffered by conventional microscopes of potential incompatibilitybetween objective lenses and binoculars is immediately eliminated. Oneof ordinary skill will similarly realize that the problem of achievingparfocality between the eyepieces of the microscope and a digital imageon a display monitor is also eliminated by virtue of not having anyeyepieces. Since scanner 11 also overcomes the field of view limitationof a traditional microscope by providing a field of view that ispractically limited only by the physical boundaries of sample 120, theimportance of magnification in an all-digital imaging microscope such asprovided by scanner 11 is limited. Once a portion of sample 120 has beendigitized, it is straightforward to apply electronic magnification,sometimes known as electric zoom, to an image of sample 120 in order toincrease its magnification. Increasing the magnification of an imageelectronically has the effect of increasing the size of that image onthe monitor that is used to display the image. If too much electroniczoom is applied, then the display monitor will be able to show onlyportions of the magnified image. However, it is not possible to useelectronic magnification to display information that was not present inthe original optical signal that was digitized in the first place. Sinceone of the objectives of scanner 11, in an embodiment, is to providehigh quality digital images, in lieu of visual observation through theeyepieces of a microscope, the content of the images acquired by scanner11 should include as much image detail as possible. The term resolutionis typically used to describe such image detail and the termdiffraction-limited is used to describe the wavelength-limited maximumspatial detail available in an optical signal. Scanner 11 providesdiffraction-limited digital imaging by selection of a tube lens focallength that is matched according to the well-known Nyquist samplingcriteria to both the size of an individual pixel element in alight-sensing camera such as line scan camera 18 and to the numericalaperture of microscope objective lens 130. It is well known thatnumerical aperture, not magnification, is the resolution-limitingattribute of a microscope objective lens.

An example will help to illustrate the optimum selection of a tube lensfocal length that is part of line scan camera focusing optics 34.Consider again the 20× microscope objective lens 130 with 9 millimeterfocal length discussed previously, and assume that this objective lenshas a numerical aperture of 0.50. Assuming no appreciable degradationfrom the condenser, the diffraction-limited resolving power of thisobjective lens at a wavelength of 500 nanometers is approximately 0.6micrometers, obtained using the well-known Abbe relationship. Assumefurther that line scan camera 18, which in an embodiment has a pluralityof 14 micrometer square pixels, is used to detect a portion of sample120. In accordance with sampling theory, it is necessary that at leasttwo sensor pixels subtend the smallest resolvable spatial feature. Inthis case, the tube lens must be selected to achieve a magnification of46.7, obtained by dividing 28 micrometers, which corresponds to two 14micrometer pixels, by 0.6 micrometers, the smallest resolvable featuredimension. The optimum tube lens optic focal length is therefore about420 millimeters, obtained by multiplying 46.7 by 9. Line scan focusingoptics 34 with a tube lens optic having a focal length of 420millimeters will therefore be capable of acquiring images with the bestpossible spatial resolution, similar to what would be observed byviewing a specimen under a microscope using the same 20× objective lens.To reiterate, scanner 11 utilizes a traditional 20× microscope objectivelens 130 in a higher magnification optical configuration (about 47× inthe example above) in order to acquire diffraction-limited digitalimages. If a traditional 20× magnification objective lens 130 with ahigher numerical aperture were used, say 0.75, the required tube lensoptic magnification for diffraction-limited imaging would be about 615millimeters, corresponding to an overall optical magnification of 68×.Similarly, if the numerical aperture of the 20× objective lens were only0.3, the optimum tube lens optic magnification would only be about 28×,which corresponds to a tube lens optic focal length of approximately 252millimeters. Line scan camera focusing optics 34 may be modular elementsof scanner 11 that can be interchanged as necessary for optimum digitalimaging. The advantage of diffraction-limited digital imaging isparticularly significant for applications, for example bright fieldmicroscopy, in which the reduction in signal brightness that accompaniesincreases in magnification is readily compensated by increasing theintensity of an appropriately designed illumination system 28.

In principle, it is possible to attach external magnification-increasingoptics to a conventional microscope-based digital imaging system toeffectively increase the tube lens magnification so as to achievediffraction-limited imaging as has just been described for scanner 11.However, the resulting decrease in the field of view is oftenunacceptable, making this approach impractical. Furthermore, many usersof microscopes typically do not understand enough about the details ofdiffraction-limited imaging to effectively employ these techniques ontheir own. In practice, digital cameras are attached to microscope portswith magnification-decreasing optical couplers to attempt to increasethe size of the field of view to something more similar to what can beseen through the eyepiece. The standard practice of adding de-magnifyingoptics is a step in the wrong direction if the goal is to obtaindiffraction-limited digital images.

In a conventional microscope, different power objectives lenses aretypically used to view the specimen at different resolutions andmagnifications. Standard microscopes have a nosepiece that holds fiveobjectives lenses. In an all-digital imaging system, such as scanner 11,there is a need for only one microscope objective lens 130 with anumerical aperture corresponding to the highest spatial resolutiondesirable. Thus, in an embodiment, scanner 11 consists of only onemicroscope objective lens 130. Once a diffraction-limited digital imagehas been captured at this resolution, it is straightforward usingstandard digital image processing techniques, to present imageryinformation at any desirable reduced resolutions and magnifications.

One embodiment of scanner 11 is based on a Dalsa SPARK line scan camera18 with 1024 pixels (picture elements) arranged in a linear array, witheach pixel having a dimension of 14 by 14 micrometers. Any other type oflinear array, whether packaged as part of a camera or custom-integratedinto an imaging electronic module, can also be used. The linear array inone embodiment effectively provides eight bits of quantization, butother arrays providing higher or lower level of quantization may also beused. Alternate arrays based on three-channel red-green-blue (RGB) colorinformation or time delay integration (TDI), may also be used. TDIarrays provide a substantially better signal-to-noise ratio (SNR) in theoutput signal by summing intensity data from previously imaged regionsof a specimen, yielding an increase in the SNR that is in proportion tothe square-root of the number of integration stages. TDI arrays cancomprise multiple stages of linear arrays. TDI arrays are available with24, 32, 48, 64, 96, or even more stages. Scanner 11 also supports lineararrays that are manufactured in a variety of formats including some with512 pixels, some with 1024 pixels, and others having as many as 4096pixels. Appropriate, but well known, modifications to illuminationsystem 28 and line scan camera focusing optics 34 may be required toaccommodate larger arrays. Linear arrays with a variety of pixel sizescan also be used in scanner 11. The salient requirement for theselection of any type of line scan camera 18 is that sample 120 can bein motion with respect to line scan camera 18 during the digitization ofsample 120, in order to obtain high quality images, overcoming thestatic requirements of the conventional imaging tiling approaches knownin the prior art.

The output signal of line scan camera 18 is connected to data processor21. In an embodiment, data processor 21 includes a central processingunit with ancillary electronics (e.g., a motherboard) to support atleast one signal digitizing electronics board such as an imaging boardor a frame grabber. In an embodiment, the imaging board is an EPIXPIXCID24 PCI bus imaging board. However, there are many other types ofimaging boards or frame grabbers from a variety of manufacturers whichcould be used in place of the EPIX board. An alternative embodimentcould be a line scan camera that uses an interface such as IEEE 1394,also known as Firewire, to bypass the imaging board altogether and storedata directly on data storage 38 (e.g., a hard disk).

Data processor 21 is also connected to a memory 36, such as randomaccess memory (RAM), for the short-term storage of data, and to datastorage 38, such as a hard drive, for long-term data storage. Further,data processor 21 is connected to a communications port 40 that isconnected to a network 42 such as a local area network (LAN), a widearea network (WAN), a metropolitan area network (MAN), an intranet, anextranet, or the Internet. Memory 36 and data storage 38 are alsoconnected to each other. Data processor 21 is also capable of executingcomputer programs, in the form of software, to control critical elementsof scanner 11 such as line scan camera 18 and stage controller 22, orfor a variety of image-processing functions, image-analysis functions,or networking. Data processor 21 can be based on any operating system,including operating systems such as Windows, Linux, OS/2, Mac OS, andUnix. In an embodiment, data processor 21 operates based on the WindowsNT operating system.

Data processor 21, memory 36, data storage 38, and communication port 40are each elements that can be found in a conventional computer. Oneexample would be a personal computer such as a Dell Dimension XPS T500that features a Pentium III 500 MHz processor and up to 756 megabytes(MB) of RAM. In an embodiment, the computer elements which include thedata processor 21, memory 36, data storage 38, and communications port40 are all internal to scanner 11, so that the only connection ofscanner 11 to the other elements of system 10 is via communication port40. In an alternative embodiment of scanner 11, the computer elementscould be external to scanner 11 with a corresponding connection betweenthe computer elements and scanner 11.

In an embodiment, scanner 11 integrates optical microscopy, digitalimaging, motorized sample positioning, computing, and network-basedcommunications into a single-enclosure unit. The major advantage ofpackaging scanner 11 as a single-enclosure unit, with communicationsport 40 as the primary means of data input and output, are reducedcomplexity and increased reliability. The various elements of scanner 11are optimized to work together, in sharp contrast to traditionalmicroscope-based imaging systems in which the microscope, light source,motorized stage, camera, and computer are typically provided bydifferent vendors and require substantial integration and maintenance.

Communication port 40 provides a means for rapid communications with theother elements of system 10, including network 42. One communicationsprotocol for communications port 40 is a carrier-sense multiple-accesscollision detection protocol such as Ethernet, together with the TCP/IPprotocol for transmission control and internetworking. Scanner 11 isintended to work with any type of transmission media, includingbroadband, baseband, coaxial cable, twisted pair, fiber optics, DSL, orwireless.

In one embodiment, control of scanner 11 and review of the imagery datacaptured by scanner 11 are performed on a computer 44 that is connectedto network 42. In an embodiment, computer 44 is connected to a displaymonitor 46 to provide imagery information to an operator. A plurality ofcomputers 44 may be connected to network 42. In an embodiment, computer44 communicates with scanner 11 using a network browser such as InternetExplorer™ from Microsoft™, Chrome™ from Google™, Safari™ from Apple™,etc. Images are stored on scanner 11 in a common compressed format, suchas JPEG, which is an image format that is compatible with standardimage-decompression methods that are already built into most commercialbrowsers. Other standard or non-standard, lossy or lossless, imagecompression formats will also work. In one embodiment, scanner 11 is aweb server providing an operator interface that is based on web pagesthat are sent from scanner 11 to computer 44. For dynamic review ofimagery data, an embodiment of scanner 11 is based on playing back, forreview on display monitor 46 that is connected to computer 44, multipleframes of imagery data using standard multiple-frame browser compatiblesoftware packages such as Media-Player™ from Microsoft™, Quicktime™ fromApple™, or RealPlayer™ from Real Networks™. In an embodiment, thebrowser on computer 44 uses the Hypertext Transfer Protocol (HTTP)together with TCP for transmission control.

There are, and will be in the future, many different means and protocolsby which scanner 11 could communicate with computer 44, or a pluralityof computers. While one embodiment is based on standard means andprotocols, the approach of developing one or multiple customizedsoftware modules known as applets is equally feasible and may bedesirable for selected future applications of scanner 11. Furthermore,there are no constraints that computer 44 be of any specific type suchas a personal computer (PC) or be manufactured by any specific companysuch as Dell™. One of the advantages of a standardized communicationsport 40 is that any type of computer 44, operating common networkbrowser software, can communicate with scanner 11.

If desired, it is possible, with some modifications to scanner 11, toobtain spectrally-resolved images. Spectrally-resolved images are imagesin which spectral information is measured at every image pixel.Spectrally-resolved images could be obtained by replacing line scancamera 18 of scanner 11 with an optical slit and an imagingspectrograph. The imaging spectrograph uses a two-dimensional CCDdetector to capture wavelength-specific intensity data for a column ofimage pixels by using a prism or grating to disperse the optical signalthat is focused on the optical slit along each of the rows of thedetector.

FIG. 14B is a block diagram of a second embodiment of an opticalmicroscopy system 10, according to an embodiment. In this system 10,scanner 11 is more complex and expensive than the embodiment shown inFIG. 14A. The additional attributes of scanner 11 that are shown do notall have to be present for any alternative embodiment to functioncorrectly. FIG. 14B is intended to provide a reasonable example ofadditional features and capabilities that could be incorporated intoscanner 11.

The alternative embodiment of FIG. 14B provides for a much greater levelof automation than the embodiment of FIG. 14A. A more complete level ofautomation of illumination system 28 is achieved by connections betweendata processor 21 and both light source 31 and illumination optics 32 ofillumination system 28. The connection to light source 31 may controlthe voltage, or current, in an open or closed loop fashion, in order tocontrol the intensity of light source 31. Recall that light source 31may be a halogen bulb. The connection between data processor 21 andillumination optics 32 could provide closed-loop control of the fieldiris aperture and the condenser iris to provide a means for ensuringthat optimum Köhler illumination is maintained.

Use of scanner 11 for fluorescence imaging requires easily recognizedmodifications to light source 31, illumination optics 32, and microscopeobjective lens 130. The embodiment of FIG. 14B also provides for afluorescence filter cube 50 that includes an excitation filter, adichroic filter, and a barrier filter. Fluorescence filter cube 50 ispositioned in the infinity-corrected beam path that exists betweenmicroscope objective lens 130 and line scan camera focusing optics 34.An embodiment for fluorescence imaging could include the addition of afilter wheel or tunable filter into illumination optics 32 to provideappropriate spectral excitation for the variety of fluorescent dyes ornano-crystals available on the market.

The addition of at least one beam splitter 52 into the imaging pathallows the optical signal to be split into at least two paths. Theprimary path is via line scan camera focusing optics 34, as discussedpreviously, to enable diffraction-limited imaging by line scan camera 18(which may include imaging sensor 20). A second path is provided via anarea scan camera focusing optics 54 for imaging by an area scan camera56. It should be readily apparent that proper selection of these twofocusing optics can ensure diffraction-limited imaging by the two camerasensors having different pixel sizes. Area scan camera 56 can be one ofmany types that are currently available, including a simple color videocamera, a high performance, cooled, CCD camera, or a variableintegration-time fast frame camera. Area scan camera 56 provides atraditional imaging system configuration for scanner 11. Area scancamera 56 is connected to data processor 21. If two cameras are used,for example line scan camera 18 and area scan camera 56, both cameratypes could be connected to the data processor using either a singledual-purpose imaging board, two different imaging boards, or theIEEE1394 Firewire interface, in which case one or both imaging boardsmay not be needed. Other related methods of interfacing imaging sensorsto data processor 21 are also available.

While the primary interface of scanner 11 to computer 44 is via network42, there may be instances, for example a failure of network 42, whereit is beneficial to be able to connect scanner 11 directly to a localoutput device such as display monitor 58 and to also provide local inputdevices such as a keyboard and mouse 59 that are connected directly intodata processor 21 of scanner 11. In this instance, the appropriatedriver software and hardware would have to be provided as well.

The second embodiment shown in FIG. 14B also provides for a much greaterlevel of automated imaging performance. Enhanced automation of theimaging of scanner 11 can be achieved by closing the focus-control loopcomprising piezo positioner 24, piezo controller 26, and data processor21 using well-known methods of autofocus. The second embodiment alsoprovides for a motorized nose-piece 62 to accommodate several objectiveslenses. The motorized nose-piece 62 is connected to and directed by dataprocessor 21 through a nose-piece controller 64.

There are other features and capabilities of scanner 11 which could beincorporated. For example, the process of scanning sample 120 withrespect to microscope objective lens 130 that is substantiallystationary in the X-Y plane of sample 120 could be modified to comprisescanning of microscope objective lens 130 with respect to a stationarysample 120 (i.e., moving microscope objective lens 130 in an X-Y plane).Scanning sample 120, or scanning microscope objective lens 130, orscanning both sample 120 and microscope objective lens 130simultaneously, are possible embodiments of scanner 11 which can providethe same large contiguous digital image of sample 120 as discussedpreviously.

Scanner 11 also provides a general purpose platform for automating manytypes of microscope-based analyses. Illumination system 28 could bemodified from a traditional halogen lamp or arc-lamp to a laser-basedillumination system to permit scanning of sample 120 with laserexcitation. Modifications, including the incorporation of aphotomultiplier tube or other non-imaging detector, in addition to or inlieu of line scan camera 18 or area scan camera 56, could be used toprovide a means of detecting the optical signal resulting from theinteraction of the laser energy with sample 120.

Turning now to FIG. 14C, line scan camera field of view 250 comprisesthe region of sample 120 of FIG. 14A that is imaged by a multitude ofindividual pixel elements 72 that are arranged in a linear fashion intoa linear array 74. Linear array 74 of an embodiment comprises 1024 ofthe individual pixel elements 72, with each of pixel elements 72 being14 micrometers square. In an embodiment, the physical dimensions oflinear array 74 are 14.34 millimeters by 14 micrometers. Assuming, forpurposes of discussion of the operation of the scanner 11, that themagnification between sample 120 and line scan camera 18 is ten, thenline scan camera field of view 250 corresponds to a region of sample 120that has dimensions equal to 1.43 millimeters by 1.4 micrometers. Eachpixel element 72 images an area about 1.4 micrometers by 1.4micrometers.

In one embodiment of scanner 11, the scanning and digitization isperformed in a direction of travel that alternates between image strips.This type of bi-directional scanning provides for a more rapiddigitization process than uni-directional scanning, a method of scanningand digitization which requires the same direction of travel for eachimage strip.

The capabilities of line scan camera 18 (e.g., comprising imaging sensor20) and focusing sensor 30 typically determine whether scanning andfocusing can be done bi-directionally or uni-directionally.Uni-directional systems often comprise more than one linear array 74,such as a three channel color array 86 or a multi-channel TDI array 88shown in FIG. 14C. Color array 86 detects the RGB intensities requiredfor obtaining a color image. An alternative embodiment for obtainingcolor information uses a prism to split the broadband optical signalinto the three color channels. TDI array 88 could be used in analternate embodiment of scanner 11 to provide a means of increasing theeffective integration time of line scan camera 18, while maintaining afast data rate, and without significant loss in the signal-to-noiseratio of the digital imagery data.

FIG. 15 is a block diagram illustrating an example wired or wirelesssystem 1500 that may be used in connection with various embodimentsdescribed herein. For example system 1500 may be used as or inconjunction with one or more of the mechanisms, processes, methods, orfunctions described above, and may represent components of slide scanner11, such as data processor 21. System 1500 can be any processor-enableddevice that is capable of wired or wireless data communication. Othercomputer systems and/or architectures may be also used, as will be clearto those skilled in the art.

System 1500 preferably includes one or more processors, such asprocessor 1510. Additional processors may be provided, such as anauxiliary processor to manage input/output, an auxiliary processor toperform floating point mathematical operations, a special-purposemicroprocessor having an architecture suitable for fast execution ofsignal processing algorithms (e.g., digital signal processor), a slaveprocessor subordinate to the main processing system (e.g., back-endprocessor), an additional microprocessor or controller for dual ormultiple processor systems, or a coprocessor. Such auxiliary processorsmay be discrete processors or may be integrated with the processor 1510.Examples of processors which may be used with system 1500 include,without limitation, the Pentium® processor, Core i7® processor, andXeon® processor, all of which are available from Intel Corporation ofSanta Clara, Calif.

Processor 1510 is preferably connected to a communication bus 1505.Communication bus 1505 may include a data channel for facilitatinginformation transfer between storage and other peripheral components ofsystem 1500. Communication bus 1505 further may provide a set of signalsused for communication with processor 1510, including a data bus,address bus, and control bus (not shown). Communication bus 1505 maycomprise any standard or non-standard bus architecture such as, forexample, bus architectures compliant with industry standard architecture(ISA), extended industry standard architecture (EISA), Micro ChannelArchitecture (MCA), peripheral component interconnect (PCI) local bus,or standards promulgated by the Institute of Electrical and ElectronicsEngineers (IEEE) including IEEE 488 general-purpose interface bus(GPIB), IEEE 696/S-100, and the like.

System 1500 preferably includes a main memory 1515 and may also includea secondary memory 1520. Main memory 1515 provides storage ofinstructions and data for programs executing on processor 1510, such asone or more of the functions and/or modules discussed above. It shouldbe understood that programs stored in the memory and executed byprocessor 1510 may be written and/or compiled according to any suitablelanguage, including without limitation C/C++, Java, JavaScript, Perl,Visual Basic, .NET, and the like. Main memory 1515 is typicallysemiconductor-based memory such as dynamic random access memory (DRAM)and/or static random access memory (SRAM). Other semiconductor-basedmemory types include, for example, synchronous dynamic random accessmemory (SDRAM), Rambus dynamic random access memory (RDRAM),ferroelectric random access memory (FRAM), and the like, including readonly memory (ROM).

Secondary memory 1520 may optionally include an internal memory 1525and/or a removable medium 1530. Removable medium 1530 is read fromand/or written to in any well-known manner. Removable storage medium1530 may be, for example, a magnetic tape drive, a compact disc (CD)drive, a digital versatile disc (DVD) drive, other optical drive, aflash memory drive, etc.

Removable storage medium 1530 is a non-transitory computer-readablemedium having stored thereon computer-executable code (i.e., software)and/or data. The computer software or data stored on removable storagemedium 1530 is read into system 1500 for execution by processor 1510.

In alternative embodiments, secondary memory 1520 may include othersimilar means for allowing computer programs or other data orinstructions to be loaded into system 1500. Such means may include, forexample, an external storage medium 1545 and a communication interface1540 (e.g., communication port 40), which allows software and data to betransferred from external storage medium 1545 to system 1500. Examplesof external storage medium 1545 may include an external hard disk drive,an external optical drive, an external magneto-optical drive, etc. Otherexamples of secondary memory 1520 may include semiconductor-based memorysuch as programmable read-only memory (PROM), erasable programmableread-only memory (EPROM), electrically erasable read-only memory(EEPROM), or flash memory (block-oriented memory similar to EEPROM).

As mentioned above, system 1500 may include a communication interface1540. Communication interface 1540 allows software and data to betransferred between system 1500 and external devices (e.g. printers),networks, or other information sources. For example, computer softwareor executable code may be transferred to system 1500 from a networkserver via communication interface 1540. Examples of communicationinterface 1540 include a built-in network adapter, network interfacecard (NIC), Personal Computer Memory Card International Association(PCMCIA) network card, card bus network adapter, wireless networkadapter, Universal Serial Bus (USB) network adapter, modem, a networkinterface card (NIC), a wireless data card, a communications port, aninfrared interface, an IEEE 1394 fire-wire, or any other device capableof interfacing system 550 with a network or another computing device.Communication interface 1540 preferably implements industry-promulgatedprotocol standards, such as Ethernet IEEE 802 standards, Fiber Channel,digital subscriber line (DSL), asynchronous digital subscriber line(ADSL), frame relay, asynchronous transfer mode (ATM), integrateddigital services network (ISDN), personal communications services (PCS),transmission control protocol/Internet protocol (TCP/IP), serial lineInternet protocol/point to point protocol (SLIP/PPP), and so on, but mayalso implement customized or non-standard interface protocols as well.

Software and data transferred via communication interface 1540 aregenerally in the form of electrical communication signals 1555. Thesesignals 1555 may be provided to communication interface 1540 via acommunication channel 1550. In an embodiment, communication channel 1550may be a wired or wireless network, or any variety of othercommunication links. Communication channel 1550 carries signals 1555 andcan be implemented using a variety of wired or wireless communicationmeans including wire or cable, fiber optics, conventional phone line,cellular phone link, wireless data communication link, radio frequency(“RF”) link, or infrared link, just to name a few.

Computer-executable code (i.e., computer programs or software) is storedin main memory 1515 and/or the secondary memory 1520. Computer programscan also be received via communication interface 1540 and stored in mainmemory 1515 and/or secondary memory 1520. Such computer programs, whenexecuted, enable system 1500 to perform the various functions of thedisclosed embodiments as described elsewhere herein.

In this description, the term “computer-readable medium” is used torefer to any non-transitory computer-readable storage media used toprovide computer-executable code (e.g., software and computer programs)to system 1500. Examples of such media include main memory 1515,secondary memory 1520 (including internal memory 1525, removable medium1530, and external storage medium 1545), and any peripheral devicecommunicatively coupled with communication interface 1540 (including anetwork information server or other network device). Thesenon-transitory computer-readable mediums are means for providingexecutable code, programming instructions, and software to system 1500.

In an embodiment that is implemented using software, the software may bestored on a computer-readable medium and loaded into system 1500 by wayof removable medium 1530, I/O interface 1535, or communication interface1540. In such an embodiment, the software is loaded into system 1500 inthe form of electrical communication signals 1555. The software, whenexecuted by processor 1510, preferably causes processor 1510 to performthe features and functions described elsewhere herein.

In an embodiment, I/O interface 1535 provides an interface between oneor more components of system 1500 and one or more input and/or outputdevices. Example input devices include, without limitation, keyboards,touch screens or other touch-sensitive devices, biometric sensingdevices, computer mice, trackballs, pen-based pointing devices, and thelike. Examples of output devices include, without limitation, cathoderay tubes (CRTs), plasma displays, light-emitting diode (LED) displays,liquid crystal displays (LCDs), printers, vacuum florescent displays(VFDs), surface-conduction electron-emitter displays (SEDs), fieldemission displays (FEDs), and the like.

System 1500 also includes optional wireless communication componentsthat facilitate wireless communication over a voice network and/or adata network. The wireless communication components comprise an antennasystem 1570, a radio system 1565, and a baseband system 1560. In system1500, radio frequency (RF) signals are transmitted and received over theair by antenna system 1570 under the management of radio system 1565.

In one embodiment, antenna system 1570 may comprise one or more antennaeand one or more multiplexors (not shown) that perform a switchingfunction to provide antenna system 1570 with transmit and receive signalpaths. In the receive path, received RF signals can be coupled from amultiplexor to a low noise amplifier (not shown) that amplifies thereceived RF signal and sends the amplified signal to radio system 1565.

In alternative embodiments, radio system 1565 may comprise one or moreradios that are configured to communicate over various frequencies. Inan embodiment, radio system 1565 may combine a demodulator (not shown)and modulator (not shown) in one integrated circuit (IC). Thedemodulator and modulator can also be separate components. In theincoming path, the demodulator strips away the RF carrier signal leavinga baseband receive audio signal, which is sent from radio system 1565 tobaseband system 1560.

If the received signal contains audio information, then baseband system1560 decodes the signal and converts it to an analog signal. Then thesignal is amplified and sent to a speaker. Baseband system 1560 alsoreceives analog audio signals from a microphone. These analog audiosignals are converted to digital signals and encoded by baseband system1560. Baseband system 1560 also codes the digital signals fortransmission and generates a baseband transmit audio signal that isrouted to the modulator portion of radio system 1565. The modulatormixes the baseband transmit audio signal with an RF carrier signalgenerating an RF transmit signal that is routed to antenna system 1570and may pass through a power amplifier (not shown). The power amplifieramplifies the RF transmit signal and routes it to antenna system 1570where the signal is switched to the antenna port for transmission.

Baseband system 1560 is also communicatively coupled with processor1510, which may be a central processing unit (CPU). Processor 1510 hasaccess to data storage areas 1515 and 1520. Processor 1510 is preferablyconfigured to execute instructions (i.e., computer programs or software)that can be stored in main memory 1515 or secondary memory 1520.Computer programs can also be received from baseband processor 1560 andstored in main memory 1510 or in secondary memory 1520, or executed uponreceipt. Such computer programs, when executed, enable system 1500 toperform the various functions of the disclosed embodiments. For example,data storage areas 1515 or 1520 may include various software modules.

Those of skill in the art will appreciate that the various illustrativelogical blocks, modules, circuits, and method steps described inconnection with the above described figures and the embodimentsdisclosed herein can often be implemented as electronic hardware,computer software, or combinations of both. To clearly illustrate thisinterchangeability of hardware and software, various illustrativecomponents, blocks, modules, circuits, and steps have been describedabove generally in terms of their functionality. Whether suchfunctionality is implemented as hardware or software depends upon theparticular application and design constraints imposed on the overallsystem. Skilled persons can implement the described functionality invarying ways for each particular application, but such implementationdecisions should not be interpreted as causing a departure from thescope of the invention. In addition, the grouping of functions within amodule, block, circuit, or step is for ease of description. Specificfunctions or steps can be moved from one module, block, or circuit toanother without departing from the invention.

Any of the software components described herein may take a variety offorms. For example, a component may be a stand-alone software package,or it may be a software package incorporated as a “tool” in a largersoftware product. It may be downloadable from a network, for example, awebsite, as a stand-alone product or as an add-in package forinstallation in an existing software application. It may also beavailable as a client-server software application, as a web-enabledsoftware application, and/or as a mobile application.

The above description of the disclosed embodiments is provided to enableany person skilled in the art to make or use the subject matter of thepresent application. Various modifications to these embodiments will bereadily apparent to those skilled in the art, and the general principlesdescribed herein can be applied to other embodiments without departingfrom the spirit or scope of the present application. Thus, it is to beunderstood that the description and drawings presented herein representa presently preferred embodiment and are therefore representative of thesubject matter which is broadly contemplated by the present application.It is further understood that the scope of the present application fullyencompasses other embodiments that may become obvious to those skilledin the art and that the scope of the present application is accordinglynot limited.

What is claimed is:
 1. A system for scanning a sample to acquire adigital image of the sample, the system comprising: an objective lenshaving an optical axis that is orthogonal to a surface of a stage; animaging sensor located above the objective lens; a focusing sensorhaving one point that is parfocal with the imaging sensor, the focusingsensor including a first region and a second region different from thefirst region; optical components arranged between the objective lens andthe focusing sensor and configured to form a focusing image of a sampleon the first region of the focusing sensor and a mirror image of thefocusing image on the second region of the focusing sensor; and at leastone hardware processor operationally coupled to the imaging sensor andthe focusing sensor and configured to: acquire the focusing image andthe mirror image of the focusing image from the focusing sensor, acquirean image of the sample from the imaging sensor, and determine adirection for moving the objective lens based on a relationship betweenthe focusing image and the mirror image.
 2. The system of claim 1,wherein the at least one hardware processor is further configured toacquire a third focusing image of the sample from a region of thefocusing sensor different from the first and second regions of thefocusing sensor.
 3. The system of claim 1, wherein the at least onehardware processor is further configured to: move the objective lensalong the optical axis until a minimum or maximum point of a contrastfunction corresponds to a parfocal point on one or both of the focusingimage and the mirror image of the focusing sensor, wherein the contrastfunction comprises a relationship between a contrast measure of thefocusing image and a contrast measure of the mirror image, and whereinthe parfocal point is parfocal with the imaging sensor, wherein therelationship is a ratio of the contrast measure of the focusing image tothe contrast measure of the mirror image.
 4. The system of claim 1,wherein the focusing image comprises pixels acquired at decreasing focaldistances in a direction from a first side of the sample to a secondside of the sample, and wherein the mirror image comprises pixelsacquired at increasing focal distances in the direction from the firstside of the sample to the second side of the sample.
 5. The system ofclaim 1, wherein the optical components comprise: at least one secondbeam splitter optically coupled to the objective lens and configured tosplit a light beam from the objective lens into at least a first path toa first region of the focusing sensor and a second path to a secondregion of the focusing sensor; and at least one optical element thatreverses the light beam along the second path.
 6. The system of claim 5,wherein the at least one optical element comprises a dove prism.
 7. Thesystem of claim 5, wherein the at least one optical element comprises atleast one mirror.
 8. The system of claim 1, wherein the focusing sensoris tilted with respect to an optical path between the objective lens andthe focusing sensor.
 9. The system of claim 1, wherein the focusingsensor is orthogonal to an optical path between the objective lens andthe focusing sensor.
 10. The system of claim 1, wherein the at least onehardware processor is configured to, for each portion of the sample tobe scanned, acquire the focusing image and the mirror image during aretrace process of a line scanning process.
 11. A method for scanning aslide, comprising: acquire a focusing image of a sample and a mirrorimage of the focusing image from a focusing sensor, the focusing sensorincluding a first region and a second region different from the firstregion, the focusing image having one point that is parfocal with animaging sensor, wherein optical components are arranged between anobjective lens and the focusing sensor to form the focusing image on thefirst region of the focusing sensor and the mirror image on the secondregion of the focusing sensor; acquire an image of the sample from theimaging sensor; and determine a direction for moving the objective lensbased on a relationship between the focusing image and the mirror image.12. The method of claim 11, further comprising: acquiring a thirdfocusing image of the sample from a region of the focusing sensordifferent from the first and second regions of the focusing sensor. 13.The method of claim 11, further comprising: moving the objective lensalong an optical axis until a minimum or maximum point of a contrastfunction corresponds to a parfocal point on one or both of the focusingimage and the mirror image of the focusing sensor, wherein the contrastfunction comprises a relationship between a contrast measure of thefocusing image and a contrast measure of the mirror image, and whereinthe parfocal point is parfocal with the imaging sensor.
 14. The methodof claim 11, wherein, for each portion of the sample to be scanned, theacquisition of the focusing image and the mirror image is performedduring a retrace process of a line scanning process.
 15. Anon-transitory computer-readable medium having stored thereoninstructions which, when executed by a hardware processor, cause thehardware processor to: acquire a focusing image and a mirror image ofthe focusing image from a focusing sensor of a digital image scanningsystem, the system including an objective lens having an optical axisthat is orthogonal to a surface of a stage, an imaging sensor locatedabove the objective lens, the focusing sensor having one point that isparfocal with the imaging sensor, the focusing sensor including a firstregion and a second region different from the first region, and opticalcomponents arranged between the objective lens and the focusing sensorand configured to form a focusing image of a sample on the first regionof the focusing sensor and a mirror image of the focusing image on thesecond region of the focusing sensor; acquire an image of the samplefrom the imaging sensor; and determine a direction for moving theobjective lens based on a relationship between the focusing image andthe mirror image.
 16. The non-transitory computer-readable medium ofclaim 15, wherein the instructions are further configured to cause theprocessor to: acquire a third focusing image of the sample from a regionof the focusing sensor different from the first and second regions ofthe focusing sensor.
 17. The non-transitory computer-readable medium ofclaim 15, wherein the instructions are further configured to cause theprocessor to: move the objective lens along the optical axis until aminimum or maximum point of a contrast function corresponds to aparfocal point on one or both of the focusing image and the mirror imageof the focusing sensor, wherein the contrast function comprises arelationship between a contrast measure of the focusing image and acontrast measure of the mirror image, and wherein the parfocal point isparfocal with the imaging sensor, wherein the relationship is a ratio ofthe contrast measure of the focusing image to the contrast measure ofthe mirror image.
 18. The non-transitory computer-readable medium ofclaim 15, wherein the focusing image comprises pixels acquired atdecreasing focal distances in a direction from a first side of thesample to a second side of the sample, and wherein the mirror imagecomprises pixels acquired at increasing focal distances in the directionfrom the first side of the sample to the second side of the sample. 19.The non-transitory computer-readable medium of claim 15, wherein theoptical components comprise: at least one second beam splitter opticallycoupled to the objective lens and configured to split a light beam fromthe objective lens into at least a first path to a first region of thefocusing sensor and a second path to a second region of the focusingsensor; and at least one optical element that reverses the light beamalong the second path.
 20. A system for scanning a sample to acquire adigital image of the sample, the system comprising: an objective lenshaving an optical axis that is orthogonal to a surface of a stage; animaging sensor located above the objective lens, wherein a first portionof the imaging sensor is configured to acquire a main image and a secondportion of the imaging sensor is configured to acquire a focusing image;optical components arranged between the objective lens and the imagingsensor and configured to form the focusing image of a sample on a firstregion of the second portion of the imaging sensor and a mirror image ofthe focusing image on a second region of the second portion of theimaging sensor; and at least one hardware processor operationallycoupled to the imaging sensor and configured to: acquire the focusingimage and the mirror image of the focusing image from the imagingsensor, acquire the main image of the sample from the imaging sensor,and determine a direction for moving the objective lens based on arelationship between the focusing image and the mirror image.
 21. Thesystem of claim 20, wherein the at least one hardware processor isfurther configured to: move the objective lens along the optical axisuntil a minimum or maximum point of a contrast function corresponds to aparfocal point on one or both of the focusing image and the mirror imageof the focusing sensor, wherein the contrast function comprises arelationship between a contrast measure of the focusing image and acontrast measure of the mirror image, and wherein the parfocal point isparfocal with the imaging sensor, wherein the relationship is a ratio ofthe contrast measure of the focusing image to the contrast measure ofthe mirror image.
 22. The system of claim 20, wherein the opticalcomponents comprise: at least one second beam splitter optically coupledto the objective lens and configured to split a light beam from theobjective lens into at least a first path to a first region of thefocusing sensor and a second path to a second region of the focusingsensor; and at least one optical element that reverses the light beamalong the second path.